Binding molecules capable of neutralizing rabies virus and uses thereof

ABSTRACT

Provided are binding molecules that specifically bind to rabies virus and are capable of neutralizing the virus. Further provided are nucleic acid molecules encoding the binding molecules, compositions comprising the binding molecules and methods of identifying or producing the binding molecules. The binding molecules can be used in the diagnosis, prophylaxis and/or treatment of a condition resulting from rabies virus. In certain embodiments, they can be used in the post-exposure prophylaxis of rabies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/590,126, filed Oct. 31, 2006, now U.S. Pat. No. 7,579,446, whichapplication is a continuation of International Patent Appln. No.PCT/EP2005/052410 filed May 26, 2005, and published in English as PCTInternat'l Publication No. WO 2005/118644 A2, on Dec. 15, 2005, whichapplication claims priority to Internat'l Patent Appln. No.PCT/EP2005/050953 filed Mar. 3, 2005, which application claims priorityto Internat'l Patent Appln. No. PCT/EP2005/050310, filed Jan. 25, 2005,which application claims priority to Internat'l Patent Appln. No.PCT/EP2004/052772 filed Nov. 3, 2004, which application claims priorityto Internat'l Patent Appln. No. PCT/EP2004/052286 filed Sep. 23, 2004,which application claims priority to Internat'l Patent Appln. No.PCT/EP2004/051661 filed Jul. 29, 2004 which application claims priorityto Internat'l Patent Appln. No. PCT/EP2004/050943 filed May 27, 2004,and U.S. Provisional Patent Appln. Ser. No. 60/575,023 filed May 27,2004, the contents of the entirety of each of which are incorporatedherein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5) Sequence Listing SubmittedOn Compact Disc

Pursuant to 37 C.F.R. §1.52(e)(1)(ii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated herein by reference. A second compact disc is beingsubmitted herewith and is an identical copy of the first compact disc.The discs are labeled “copy 1” and “copy 2,” respectively, and each disccontains one file entitled “Sequence Listing 2578-7990US.txt” which is337 KB and was created on Oct. 30, 2006.

TECHNICAL FIELD

The invention relates generally to biotechnology and medicine. Inparticular, the invention relates to binding molecules directed againstrabies, such as virus-neutralizing binding molecules. The bindingmolecules are useful in the post-exposure prophylaxis of rabies.

BACKGROUND

Rabies is a viral infection with nearly worldwide distribution thataffects principally wild and domestic animals but also involves humans,resulting in a devastating, almost invariably fatal encephalitis.Annually, more than 70,000 human fatalities are estimated, and millionsof others require post-exposure treatment.

The rabies virus is a bullet-shaped, enveloped, single-stranded RNAvirus classified in the rhabdovirus family and Lyssavirus genus. Thegenome of rabies virus codes for five viral proteins: RNA-dependent RNApolymerase (L); a nucleoprotein (N); a phosphorylated protein (P); amatrix protein (M) located on the inner side of the viral proteinenvelope; and an external surface glycoprotein (G).

The G protein (62-67 kDa) is a type-I glycoprotein composed of 505 aminoacids that has two to four potential N-glycosylation sites, of whichonly one or two are glycosylated depending on the virus strains. The Gprotein forms the protrusions that cover the outer surface of the virionenvelope and is known to induce virus-neutralizing antibodies.

Rabies can be treated or prevented by both passive and activeimmunizations. Rabies post-exposure prophylaxis includes prompt localwound care and administration of both passive (anti-rabiesimmunoglobulins) and active (vaccines) immunizations.

Currently, the anti-rabies immunoglobulins (RIG) are prepared from theserum samples of either rabies virus-immune humans (HRIG) or rabiesvirus-immune horses (ERIG). A disadvantage of ERIG as well as HRIG isthat they are not available in sufficient amounts and, in case of HRIG,are too expensive. In addition, the use of ERIG might lead to adversereactions such as anaphylactic shock. The possibility of contaminationby known or unknown pathogens is an additional concern associated withHRIG. To overcome these disadvantages it has been suggested to usemonoclonal antibodies capable of neutralizing rabies virus inpost-exposure prophylaxis. Rabies virus-neutralizing murine monoclonalantibodies are known in the art (see, Schumacher et al., 1989). However,the use of murine antibodies in vivo is limited due to problemsassociated with administration of murine antibodies to humans, such asshort serum half life, an inability to trigger certain human effectorfunctions and elicitation of an unwanted dramatic immune responseagainst the murine antibody in a human (the “human anti-mouse antibody”(HAMA) reaction).

Recently, human rabies virus-neutralizing monoclonal antibodies havebeen described (see, Dietzschold et al., 1990, Champion et al., 2000,and Hanlon et al., 2001). For human anti-rabies monoclonal antibodies tobe as effective as HRIG in post-exposure prophylaxis a mixture ofmonoclonal antibodies should be used. In such a mixture each antibodyshould bind to a different epitope or site on the virus to prevent theescape of resistant variants of the virus.

Currently, a significant need still exists for new human rabiesvirus-neutralizing monoclonal antibodies having improved post-exposureprophylactic potential, particularly antibodies having differentepitope-recognition specificities.

SUMMARY OF THE INVENTION

Described are human monoclonal antibodies that offer the potential to beused in mixtures useful in the post-exposure prophylaxis of a wide rangeof rabies viruses and neutralization-resistant variants thereof.

Herebelow follow definitions of terms as used herein.

Definitions

Binding molecule: As used herein the term “binding molecule” refers toan intact immunoglobulin including monoclonal antibodies, such aschimeric, humanized or human monoclonal antibodies, or to anantigen-binding and/or variable domain comprising fragment of animmunoglobulin that competes with the intact immunoglobulin for specificbinding to the binding partner of the immunoglobulin, e.g., rabies virusor a fragment thereof such as, for instance, the G protein. Regardlessof structure, the antigen-binding fragment binds with the same antigenthat is recognized by the intact immunoglobulin. An antigen-bindingfragment can comprise a peptide or polypeptide comprising an amino acidsequence of at least two contiguous amino acid residues, at least fivecontiguous amino acid residues, at least ten contiguous amino acidresidues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 30 contiguous amino acid residues, at least 35contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least 80 contiguous amino acid residues, at least 90 contiguous aminoacid residues, at least 100 contiguous amino acid residues, at least 125contiguous amino acid residues, at least 150 contiguous amino acidresidues, at least 175 contiguous amino acid residues, at least 200contiguous amino acid residues, or at least 250 contiguous amino acidresidues of the amino acid sequence of the binding molecule.

The term “binding molecule,” as used herein includes all immunoglobulinclasses and subclasses known in the art. Depending on the amino acidsequence of the constant domain of their heavy chains, binding moleculescan be divided into the five major classes of intact antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.

Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)₂, Fv,dAb, Fd, complementarity determining region (CDR) fragments,single-chain antibodies (scFv), bivalent single-chain antibodies,single-chain phage antibodies, diabodies, triabodies, tetrabodies,(poly)peptides that contain at least a fragment of an immunoglobulinthat is sufficient to confer specific antigen binding to the(poly)peptide, etc. The above fragments may be produced synthetically orby enzymatic or chemical cleavage of intact immunoglobulins or they maybe genetically engineered by recombinant DNA techniques. The methods ofproduction are well known in the art and are described, for example, in“Antibodies: A Laboratory Manual,” edited by E. Harlow and D. Lane(1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., whichis incorporated herein by reference. A binding molecule orantigen-binding fragment thereof may have one or more binding sites. Ifthere is more than one binding site, the binding sites may be identicalto one another or they may be different.

The binding molecule can be a naked or unconjugated binding molecule butcan also be part of an immunoconjugate. A naked or unconjugated bindingmolecule is intended to refer to a binding molecule that is notconjugated, operatively linked or otherwise physically or functionallyassociated with an effector moiety or tag, such as inter alia a toxicsubstance, a radioactive substance, a liposome, or an enzyme. It will beunderstood that naked or unconjugated binding molecules do not excludebinding molecules that have been stabilized, multimerized, humanized orin any other way manipulated, other than by the attachment of aneffector moiety or tag. Accordingly, all post-translationally modifiednaked and unconjugated binding molecules are included herewith,including where the modifications are made in the natural bindingmolecule-producing cell environment, by a recombinant-bindingmolecule-producing cell, and are introduced by the hand of man afterinitial binding molecule preparation. Of course, the term naked orunconjugated binding molecule does not exclude the ability of thebinding molecule to form functional associations with effector cellsand/or molecules after administration to the body, as some of suchinteractions are necessary in order to exert a biological effect. Thelack of associated effector group or tag is therefore applied indefinition to the naked or unconjugated binding molecule in vitro, notin vivo.

Complementarity determining regions (CDR): The term “complementaritydetermining regions” as used herein means sequences within the variableregions of binding molecules, such as immunoglobulins, that usuallycontribute to a large extent to the antigen-binding site which iscomplementary in shape and charge distribution to the epitope recognizedon the antigen. The CDR regions can be specific for linear epitopes,discontinuous epitopes, or conformational epitopes of proteins orprotein fragments, either as present on the protein in its nativeconformation or, in some cases, as present on the proteins as denatured,e.g., by solubilization in SDS. Epitopes may also consist ofpost-translational modifications of proteins.

Functional variant: The term “functional variant,” as used herein,refers to a binding molecule that comprises a nucleotide and/or aminoacid sequence that is altered by one or more nucleotides and/or aminoacids compared to the nucleotide and/or amino acid sequences of theparent binding molecule and that is still capable of competing forbinding to the binding partner, e.g., rabies virus or a fragmentthereof, with the parent binding molecule. In other words, themodifications in the amino acid and/or nucleotide sequence of the parentbinding molecule do not significantly affect or alter the bindingcharacteristics of the binding molecule encoded by the nucleotidesequence or containing the amino acid sequence, i.e., the bindingmolecule is still able to recognize and bind its target. The functionalvariant may have conservative sequence modifications includingnucleotide and amino acid substitutions, additions and deletions. Thesemodifications can be introduced by standard techniques known in the art,such as site-directed mutagenesis and random PCR-mediated mutagenesis,and may comprise natural as well as non-natural nucleotides and aminoacids.

Conservative amino acid substitutions include the ones in which theamino acid residue is replaced with an amino acid residue having similarstructural or chemical properties. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.,glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan). It will be clear to the skilled artisan that otherclassifications of amino acid residue families than the one used abovecan also be employed. Furthermore, a variant may have non-conservativeamino acid substitutions, e.g., replacement of an amino acid with anamino acid residue having different structural or chemical properties.Similar minor variations may also include amino acid deletions orinsertions, or both. Guidance in determining which amino acid residuesmay be substituted, inserted, or deleted without abolishingimmunological activity may be found using computer programs well knownin the art.

A mutation in a nucleotide sequence can be a single alteration made at alocus (a point mutation), such as transition or transversion mutations,or alternatively, multiple nucleotides may be inserted, deleted orchanged at a single locus. In addition, one or more alterations may bemade at any number of loci within a nucleotide sequence. The mutationsmay be performed by any suitable method known in the art.

Host: The term “host,” as used herein, is intended to refer to anorganism or a cell into which a vector such as a cloning vector or anexpression vector has been introduced. The organism or cell can beprokaryotic or eukaryotic. It should be understood that this term isintended to refer not only to the particular subject organism or cell,but to the progeny of such an organism or cell as well. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent organism or cell, but are still included within the scopeof the term “host” as used herein.

Human: The term “human,” when applied to binding molecules as definedherein, refers to molecules that are either directly derived from ahuman or based upon a human sequence. When a binding molecule is derivedfrom or based on a human sequence and subsequently modified, it is stillto be considered human as used throughout the specification. In otherwords, the term human, when applied to binding molecules is intended toinclude binding molecules having variable and constant regions derivedfrom human germline immunoglobulin sequences based on variable orconstant regions either or not occurring in a human or human lymphocyteor in modified form. Thus, the human binding molecules may include aminoacid residues not encoded by human germline immunoglobulin sequences,comprise substitutions and/or deletions (e.g., mutations introduced by,for instance, random or site-specific mutagenesis in vitro or by somaticmutation in vivo). “Based on” as used herein refers to the situationthat a nucleic acid sequence may be exactly copied from a template, orwith minor mutations, such as by error-prone PCR methods, orsynthetically made matching the template exactly or with minormodifications. Semisynthetic molecules based on human sequences are alsoconsidered to be human as used herein.

Monoclonal antibody: The term “monoclonal antibody” as used hereinrefers to a preparation of antibody molecules of single molecularcomposition, i.e., primary structure, i.e., having a single amino acidsequence. A monoclonal antibody displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” refers to an antibody displaying a single bindingspecificity which has variable and constant regions derived from, orbased on, human germline immunoglobulin sequences or derived fromcompletely synthetic sequences. The method of preparing the monoclonalantibody is not relevant.

Nucleic acid molecule: The term “nucleic acid molecule” as used in theinvention refers to a polymeric form of nucleotides and includes bothsense and antisense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. A nucleotide refers to aribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term also includes single- and double-stranded forms ofDNA. In addition, a polynucleotide may include either or both naturallyoccurring and modified nucleotides linked together by naturallyoccurring and/or non-naturally occurring nucleotide linkages. Thenucleic acid molecules may be modified chemically or biochemically ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). The above term is also intended to include anytopological conformation, including single-stranded, double-stranded,partially duplexed, triplex, hair-pinned, circular and padlockedconformations. Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule. A reference to a nucleic acid sequence encompasses itscomplement unless otherwise specified. Thus, a reference to a nucleicacid molecule having a particular sequence should be understood toencompass its complementary strand, with its complementary sequence. Thecomplementary strand is also useful, e.g., for antisense therapy,hybridization probes and PCR primers.

Pharmaceutically acceptable excipient: By “pharmaceutically acceptableexcipient” is meant any inert substance that is combined with an activemolecule such as a drug, agent, or binding molecule for preparing anagreeable or convenient dosage form. The “pharmaceutically acceptableexcipient” is an excipient that is non-toxic, or at least of which thetoxicity is acceptable for its intended use, to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation comprising the drug, agent or bindingmolecule.

Post exposure prophylaxis: “Post exposure prophylaxis” (PEP) isindicated for persons possibly exposed to a rabid animal. Possibleexposures include bite exposure (i.e., any penetration of the skin byteeth) including animal bites, and non-bite exposure. Non-bite exposuresinclude exposure to large amounts of aerosolized rabies virus inlaboratories or caves and surgical recipients of corneas transplantedfrom patients who died of rabies. The contamination of open wounds,abrasions, mucous membranes, or theoretically, scratches, with saliva orother potentially infectious material (such as neural tissue) from arabid animal also constitutes a non-bite exposure. Other contact byitself, such as petting a rabid animal and contact with blood, urine, orfeces of a rabid animal, does not constitute an exposure and is not anindication for prophylaxis. PEP should begin as soon as possible afteran exposure. If no exposure has occurred post exposure prophylaxis isnot necessary. In all post exposure prophylaxis regimens, except forpersons previously immunized, active and passive immunizations should beused concurrently.

Specifically Binding: The term “specifically binding,” as used herein,in reference to the interaction of a binding molecule, e.g., anantibody, and its binding partner, e.g., an antigen, means that theinteraction is dependent upon the presence of a particular structure,e.g., an antigenic determinant or epitope, on the binding partner. Inother words, the antibody preferentially binds or recognizes the bindingpartner even when the binding partner is present in a mixture of othermolecules or organisms. The binding may be mediated by covalent ornon-covalent interactions or a combination of both. In yet other words,the term “specifically binding” means immunospecifically binding to anantigen or a fragment thereof and not immunospecifically binding toother antigens. A binding molecule that immunospecifically binds to anantigen may bind to other peptides or polypeptides with lower affinityas determined by, e.g., radioimmunoassays (RIA), enzyme-linkedimmunosorbent assays (ELISA), BIACORE, or other assays known in the art.Binding molecules or fragments thereof that immunospecifically bind toan antigen may be cross-reactive with related antigens. In certainembodiments, binding molecules or fragments thereof thatimmunospecifically bind to an antigen do not cross-react with otherantigens.

Therapeutically effective amount: The term “therapeutically effectiveamount” refers to an amount of the binding molecule as defined hereinthat is effective for post-exposure prophylaxis of rabies.

Vector: The term “vector” denotes a nucleic acid molecule into which asecond nucleic acid molecule can be inserted for introduction into ahost where it will be replicated, and in some cases expressed. In otherwords, a vector is capable of transporting a nucleic acid molecule towhich it has been linked. Cloning as well as expression vectors arecontemplated by the term “vector,” as used herein. Vectors include, butare not limited to, plasmids, cosmids, bacterial artificial chromosomes(BAC) and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. A vector containing a second nucleic acidmolecule is introduced into a cell, for example, by transformation,transfection, or by making use of bacterial or viral entry mechanisms.Other ways of introducing nucleic acid into cells are known, such aselectroporation or particle bombardment often used with plant cells, andthe like. The method of introducing nucleic acid into cells dependsamong other things on the type of cells, and so forth. This is notcritical to the invention. Certain vectors are capable of autonomousreplication in a host into which they are introduced (e.g., vectorshaving a bacterial origin of replication can replicate in bacteria).Other vectors can be integrated into the genome of a host uponintroduction into the host, and thereby are replicated along with thehost genome.

Provided are binding molecules capable of specifically binding to andneutralizing rabies virus. Furthermore, described are nucleic acidmolecules encoding at least the binding region of these bindingmolecules. The invention further provides for the use of the bindingmolecules of the invention in the post exposure prophylaxis of a subjectat risk of developing a condition resulting from rabies virus.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the comparison of the amino acid sequences of the rabiesvirus strain CVS-11 and E57 escape viruses. Virus-infected cells wereharvested two days post-infection and total RNA was isolated. cDNA wasgenerated and used for DNA sequencing. Regions containing mutations areshown and the mutations are indicated in bold. FIG. 1A shows thecomparison of the nucleotide sequences. Numbers above amino acidsindicate amino acid numbers from rabies virus glycoprotein includingsignal peptide. FIG. 1B shows the comparison of amino acid sequences.Schematic drawing of rabies virus glycoprotein is shown on top. Theblack box indicates the signal peptide, while the gray box indicates thetransmembrane domain. The sequences in FIG. 1 are also represented bySEQ ID NOS:130 through 141 of the incorporated SEQUENCE LISTING.

FIG. 2 shows the comparison of the amino acid sequences of the rabiesvirus strain CVS-11 and EJB escape viruses. Virus-infected cells wereharvested two days post-infection and total RNA was isolated. cDNA wasgenerated and used for DNA sequencing. Regions containing mutations areshown and the mutations are indicated in bold. FIG. 2A shows thecomparison of the nucleotide sequences. Numbers above amino acidsindicate amino acid numbers from rabies virus glycoprotein including thesignal peptide. FIG. 2B shows the comparison of amino acid sequences.Schematic drawing of rabies virus glycoprotein is shown on top. Theblack box indicates the signal peptide, while the gray box indicates thetransmembrane domain. The sequences in FIG. 2 are also represented bySEQ ID NOS:142 through 151.

FIG. 3 shows the vector PDV-C06.

FIG. 4 shows a competition ELISA of anti-rabies virus scFvs and thebiotinylated anti-rabies virus antibody called CR-57. ELISA platescoated with purified rabies virus G protein were incubated with therespective scFvs before addition of CR-57bio (0.5 μg/ml). Subsequently,CR-57bio binding was monitored in absence and presence of scFvs.

FIG. 5 shows a competition ELISA of anti-rabies virus scFvs and theanti-rabies virus antibody called CR-57. ELISA plates coated withpurified rabies virus G protein were incubated with CR-57 (1 μg/ml)before addition of excess scFvs. Subsequently, scFv binding wasmonitored in absence and presence of CR-57.

FIG. 6 shows a competition ELISA assay of anti-rabies virus G proteinIgGs and the anti-rabies virus antibody called CR-57. G protein (ERAstrain) was incubated with unlabeled IgGs (shown on the X-axis).Biotinylated CR57 (CR57bio) was added and allowed to bind to the Gprotein before visualization by means of streptavidin-HRP. ELISA signalsare shown as percentage of CR57bio binding alone.

FIG. 7 shows a competition FACS assay of anti-rabies virus G proteinIgGs and the anti-rabies virus antibody called CR-57. G protein (ERAstrain) expressing PER.C6 cells were incubated with unlabeled IgGs(shown on the X-axis). Biotinylated CR57 (CR57bio) was added and allowedto bind to the G protein expressing cells before visualization by meansof streptavidin-PE. FACS signals are shown as percentage of CR57biobinding alone.

FIG. 8 shows the comparison of the amino acid sequences of CVS-11 andE98 escape viruses. Virus-infected cells were harvested two dayspost-infection and total RNA was isolated. cDNA was generated and usedfor DNA sequencing. Region containing a point mutation is shown and themutation is indicated in bold. FIG. 8A shows the comparison of thenucleotide sequences SEQ ID NOS:745 and 746. The number above thenucleotide indicates the mutated nucleotide (indicated in bold) fromrabies virus glycoprotein open reading frame without signal peptidesequence. FIG. 8B shows the comparison of amino acid sequences SEQ IDNOS:747 and 748. The number above the amino acid indicates the mutatedamino acid (indicated in bold) from rabies virus glycoprotein withoutsignal peptide sequence.

FIG. 9 shows a phylogenetic tree of 123 rabies street viruses (123rabies virus G glycoprotein sequences, Neighbor joining,Kimura-2-parameter method, 500 bootstraps). Bold indicates virusesharboring the N>D mutation as observed in E98 escape viruses.

FIG. 10 shows neutralizing epitopes on rabies glycoprotein. A schematicdrawing of the rabies virus glycoprotein is shown depicting theantigenic sites including the novel CR57 epitope. The signal peptide (19amino acids) and transmembrane domain are indicated by black boxes.Disulfide bridges are indicated. Amino acid numbering is from the matureprotein minus the signal peptide sequence.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention encompasses binding molecules capableof specifically binding to rabies virus. In certain embodiments, thebinding molecules of the invention also have rabies virus-neutralizingactivity. In certain embodiments, the binding molecules of the inventionare human binding molecules. Alternatively, they may also be bindingmolecules of other animals. Rabies virus is part of the Lyssavirusgenus. In total, the Lyssavirus genus includes eleven genotypes: rabiesvirus (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype3), Duvenhage virus (genotype 4), European bat lyssavirus 1 (genotype5), European bat lyssavirus 2 (genotype 6), Australian bat lyssaviru(genotype 7), Aravan virus (genotype 8), Khujand virus (genotype 9),Irkut virus (genotype 10) and West Caucasian virus (genotype 11).Besides binding to rabies virus, the binding molecules of the inventionmay also be capable of binding to other genotypes of the Lyssavirusgenus. In certain embodiments, the binding molecules may also be capableof neutralizing other genotypes of the Lyssavirus genus. Furthermore,the binding molecules of the invention may even be capable of binding toand/or neutralizing viruses, other than Lyssaviruses, of the rhabdovirusfamily. This family includes the genera cytorhabdovirus, ephemerovirus,lyssavirus, nucleorhabdovirus, rhabdovirus and vesiculovirus.

The binding molecules may be capable of specifically binding to rabiesvirus in its natural form or in its inactivated/attenuated form.Inactivation of rabies virus may be performed by treatment with interalia beta-propiolactone (BPL) (White and Chappel, 1982), heating at 56°C. for more than 30 minutes, gamma irradiation, treatment withacetylethylenimine or ethylenimine or treatment with ascorbic acid andcopper sulfate for 72 hours (Madhusudana et al., 2004). General viralinactivation methods well known to the skilled artisan such as interalia pasteurization (wet heat), dry heat treatment, vapor heattreatment, treatment with low pH, treatment with organicsolvent/detergent, nanofiltration; UV light irradiation may also beused. In certain embodiments, the inactivation is performed by treatmentwith beta-propiolactone (BPL). Methods to test if rabies virus is stillinfective or partly or completely inactivated are well known to theperson skilled in the art and can among others be found in “Laboratorytechniques in rabies,” edited by F. -X. Meslin, M. M. Kaplan and H.Koprowski (1996), 4th edition, Chapter 36, World Health Organization,Geneva.

The binding molecules may also be capable of specifically binding to oneor more fragments of the rabies virus such as inter alia a preparationof one or more proteins and/or (poly)peptides derived from rabies virusor a cell transfected with a rabies virus protein and/or (poly)peptide.For methods of treatment and/or prevention such as methods for postexposure prophylaxis of rabies virus the binding molecules arepreferably capable of specifically binding to surface accessibleproteins of rabies virus such as the M (see, Ameyama et al. 2003) or Gprotein. For diagnostic purposes, the human binding molecules may alsobe capable of specifically binding to proteins not present on thesurface of rabies virus. The amino acid sequence of surface accessibleand internal proteins of various known strains of rabies virus can befound in the EMBL-database and/or other databases.

In certain embodiments, the fragment at least comprises an antigenicdeterminant recognized by the human binding molecules of the invention.An “antigenic determinant” as used herein is a moiety, such as a rabiesvirus (poly)peptide, (glyco)protein, or analog or fragment thereof, thatis capable of binding to a human binding molecule of the invention withsufficiently high affinity to form a detectable antigen-binding moleculecomplex.

The binding molecules according to the invention can be intactimmunoglobulin molecules such as polyclonal or monoclonal antibodies, inparticular human monoclonal antibodies, or the binding molecules can beantigen-binding fragments including, but not limited to, Fab, F(ab′),F(ab′)₂, Fv, dAb, Fd, complementarity determining region (CDR)fragments, single-chain antibodies (scFv), bivalent single-chainantibodies, single-chain phage antibodies, diabodies, triabodies,tetrabodies, and (poly)peptides that contain at least a fragment of animmunoglobulin that is sufficient to confer specific antigen binding tothe rabies virus or fragment thereof. The binding molecules of theinvention can be used in non-isolated or isolated form. Furthermore, thebinding molecules of the invention can be used alone or in a mixturecomprising at least one human binding molecule (or variant or fragmentthereof). In other words, the binding molecules can be used incombination, e.g., as a pharmaceutical composition comprising two ormore binding molecules, variants or fragments thereof. For example,binding molecules having rabies virus-neutralizing activity can becombined in a single therapy to achieve a desired prophylactic,therapeutic or diagnostic effect.

RNA viruses such as rabies virus make use of their own RNA polymeraseduring virus replication. These RNA polymerases tend to be error-prone.This leads to the formation of so-called quasi-species during a viralinfection. Each quasi-species has a unique RNA genome, which couldresult in differences in amino acid composition of viral proteins. Ifsuch mutations occur in structural viral proteins, the virus couldpotentially escape from the host's immune system due to a change in T orB cell epitopes. The likelihood of this to happen is higher whenindividuals are treated with a mixture of two binding molecules, such ashuman monoclonal antibodies, than with a polyclonal antibody mixture(HRIG). Therefore, a prerequisite for a mixture of two human monoclonalantibodies for treatment of rabies is that the two antibodies recognizenon-overlapping, non-competing epitopes on their target antigen, i.e.,rabies virus glycoprotein. The chance of the occurrence of rabies escapeviruses is thereby minimized. As a consequence thereof, the bindingmolecules of the invention preferably are capable of reacting withdifferent, non-overlapping, non-competing epitopes of the rabies virus,such as epitopes on the rabies virus G protein. The mixture of bindingmolecules may further comprise at least one other therapeutic agent suchas a medicament suitable for the post exposure prophylaxis of rabies.

Typically, binding molecules according to the invention can bind totheir binding partners, i.e., rabies virus or fragments thereof such asrabies virus proteins, with an affinity constant (K_(d)-value) that islower than 0.2*10⁻⁴ M, 1.0*10⁻⁵ M, 1.0*10⁻⁶ M, 1.0*10⁻⁷ M, preferablylower than 1.0*10⁻⁸ M, more preferably lower than 1.0*10⁻⁹ M, morepreferably lower than 1.0*10⁻¹⁰ M, even more preferably lower than1.0*10⁻¹¹ M, and in particular lower than 1.0*10⁻¹² M. The affinityconstants can vary for antibody isotypes. For example, affinity bindingfor an IgM isotype refers to a binding affinity of at least about1.0*10⁻⁷ M. Affinity constants can for instance be measured usingsurface plasmon resonance, i.e., an optical phenomenon that allows forthe analysis of real-time biospecific interactions by detection ofalterations in protein concentrations within a biosensor matrix, forexample, using the BIACORE system (Pharmacia Biosensor AB, Uppsala,Sweden).

The binding molecules according to the invention may bind to rabiesvirus in purified/isolated or non-purified/non-isolated form. Thebinding molecules may bind to rabies virus in soluble form such as, forinstance, in a sample or may bind to rabies virus bound or attached to acarrier or substrate, e.g., microtiter plates, membranes and beads, etc.Carriers or substrates may be made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc.The surface of such supports may be solid or porous and of anyconvenient shape. Alternatively, the binding molecules may also bind tofragments of rabies virus, such as proteins or (poly)peptides of therabies virus. In certain embodiments, the binding molecules are capableof specifically binding to the rabies virus G protein or fragmentthereof. The rabies virus proteins or (poly)peptides may either be insoluble form or may bind to rabies virus bound or attached to a carrieror substrate as described above. In certain embodiments, cellstransfected with the G protein may be used as binding partner for thebinding molecules.

In certain embodiments, the binding molecules of the inventionneutralize rabies virus infectivity. This may be achieved by preventingthe attachment of rabies virus to its receptors on host cells, such asinter alia the murine p75 neurotrophin receptor, the neural celladhesion molecule (CD56) and the acetylcholine receptor, or inhibitionof the release of RNA into the cytoplasm of the cell or prevention ofRNA transcription or translation. In a specific embodiment, the bindingmolecules of the invention prevent rabies virus from infecting hostcells by at least 99%, at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to infection of hostcells by rabies virus in the absence of the binding molecules.Neutralization can, for instance, be measured as described in“Laboratory techniques in rabies,” edited by F. -X. Meslin, M. M. Kaplanand H. Koprowski (1996), 4th edition, Chapters 15-17, World HealthOrganization, Geneva. Furthermore, the human binding molecules of theinvention may be complement fixing binding molecules capable ofassisting in the lysis of enveloped rabies virus. The human bindingmolecules of the invention might also act as opsonins and augmentphagocytosis of rabies virus either by promoting its uptake via Fc orC3b receptors or by agglutinating rabies virus to make it more easilyphagocytosed.

In a preferred embodiment, the binding molecules according to theinvention comprise at least a CDR3 region comprising the amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23 and SEQ ID NO:24. In certain embodiments, the CDR3 region is aheavy chain CDR3 region.

In yet another embodiment, the binding molecules according to theinvention comprise a variable heavy chain comprising essentially anamino acid sequence selected from the group consisting of SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49. In a preferred embodiment,the binding molecules according to the invention comprise a variableheavy chain comprising essentially an amino acid sequence comprisingamino acids 1-119 of SEQ ID NO:335.

In a further embodiment, the binding molecules according to theinvention comprise a variable heavy chain comprising the amino acidsequence of SEQ ID NO:26 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:50, a variable heavy chain comprising theamino acid sequence of SEQ ID NO:27 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:51, a variable heavychain comprising the amino acid sequence of SEQ ID NO:28 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:52, avariable heavy chain comprising the amino acid sequence of SEQ ID NO:29and a variable light chain comprising the amino acid sequence of SEQ IDNO:53, a variable heavy chain comprising the amino acid sequence of SEQID NO:30 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:54, a variable heavy chain comprising the amino acidsequence of SEQ ID NO:31 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:55, a variable heavy chain comprising theamino acid sequence of SEQ ID NO:32 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:56, a variable heavychain comprising the amino acid sequence of SEQ ID NO:33 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:57, avariable heavy chain comprising the amino acid sequence of SEQ ID NO:34and a variable light chain comprising the amino acid sequence of SEQ IDNO:58, a variable heavy chain comprising the amino acid sequence of SEQID NO:35 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:59, a variable heavy chain comprising the amino acidsequence of SEQ ID NO:36 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:60, a variable heavy chain comprising theamino acid sequence of SEQ ID NO:37 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:61, a variable heavychain comprising the amino acid sequence of SEQ ID NO:38 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:62, avariable heavy chain comprising the amino acid sequence of SEQ ID NO:39and a variable light chain comprising the amino acid sequence of SEQ IDNO:63, a variable heavy chain comprising the amino acid sequence of SEQID NO:40 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:64, a variable heavy chain comprising the amino acidsequence of SEQ ID NO:41 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:65, a variable heavy chain comprising theamino acid sequence of SEQ ID NO:42 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:66, a variable heavychain comprising the amino acid sequence of SEQ ID NO:43 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:67, avariable heavy chain comprising the amino acid sequence of SEQ ID NO:44and a variable light chain comprising the amino acid sequence of SEQ IDNO:68, a variable heavy chain comprising the amino acid sequence of SEQID NO:45 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:69, a variable heavy chain comprising the amino acidsequence of SEQ ID NO:46 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:70, a variable heavy chain comprising theamino acid sequence of SEQ ID NO:47 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:71, a variable heavychain comprising the amino acid sequence of SEQ ID NO:48 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:72, avariable heavy chain comprising the amino acid sequence of SEQ ID NO:49and a variable light chain comprising the amino acid sequence of SEQ IDNO:73. In a preferred embodiment, the human binding molecules accordingto the invention comprise a variable heavy chain comprising the aminoacid sequence comprising amino acids 1-119 of SEQ ID NO:335 and avariable light chain comprising the amino acid sequence comprising aminoacids 1-107 of SEQ ID NO:337.

In a preferred embodiment, the binding molecules having rabiesvirus-neutralizing activity of the invention are administered in IgGformat, preferably IgG1 format.

Another aspect of the invention includes functional variants of bindingmolecules as defined herein. Molecules are considered to be “functionalvariants of a binding molecule according to the invention,” if thevariants are capable of competing for specific binding to rabies virusor a fragment thereof with the parent binding molecules; in other words,when the functional variants are still capable of binding to rabiesvirus or a fragment thereof. Functional variants should also still haverabies virus-neutralizing activity. Functional variants include, but arenot limited to, derivatives that are substantially similar in primarystructural sequence, but which contain e.g., in vitro or in vivomodifications, chemical and/or biochemical, that are not found in theparent binding molecule. Such modifications include inter aliaacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation,GPI-anchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, ubiquitination, and the like.

Alternatively, functional variants can be binding molecules as definedin the invention comprising an amino acid sequence containingsubstitutions, insertions, deletions or combinations thereof of one ormore amino acids compared to the amino acid sequences of the parentbinding molecules. Furthermore, functional variants can comprisetruncations of the amino acid sequence at either or both the amino orcarboxy termini. Functional variants according to the invention may havethe same or different, either higher or lower, binding affinitiescompared to the parent binding molecule but are still capable of bindingto rabies virus or a fragment thereof and are still capable ofneutralizing rabies virus. For instance, functional variants accordingto the invention may have increased or decreased binding affities forrabies virus or a fragment thereof compared to the parent bindingmolecules or may have a higher or lower rabies virus-neutralizingactivity. In certain embodiments, the amino acid sequences of thevariable regions, including, but not limited to, framework regions,hypervariable regions, in particular the CDR3 regions, are modified.Generally, the light chain and the heavy chain variable regions comprisethree hypervariable regions, comprising three CDRs, and more conservedregions, the so-called framework regions (FRs). The hypervariableregions comprise amino acid residues from CDRs and amino acid residuesfrom hypervariable loops. Functional variants intended to fall withinthe scope of the invention have at least about 50% to about 99%,preferably at least about 60% to about 99%, more preferably at leastabout 70% to about 99%, even more preferably at least about 80% to about99%, most preferably at least about 90% to about 99%, in particular atleast about 95% to about 99%, and in particular at least about 97% toabout 99% amino acid sequence homology with the parent binding moleculesas defined herein. Computer algorithms such as inter alia Gap or Bestfitknown to a person skilled in the art can be used to optimally alignamino acid sequences to be compared and to define similar or identicalamino acid residues.

In certain embodiments, functional variants may be produced when theparent binding molecule comprises a glycosylation site in its sequencethat results in glycosylation of the binding molecule upon expression ineukaryotic cells and hence might abrogate the binding to the antigen.The functional variant produced no longer contains the glycosylationsite, but will be capable of binding to rabies virus and still haveneutralizing activity.

Functional variants can be obtained by altering the parent bindingmolecules or parts thereof by general molecular biology methods known inthe art including, but not limited to, error-prone PCR,oligonucleotide-directed mutagenesis and site-directed mutagenesis.Furthermore, the functional variants may have complement fixingactivity, be capable of assisting in the lysis of enveloped rabies virusand/or act as opsonins and augment phagocytosis of rabies virus eitherby promoting its uptake via Fc or C3b receptors or by agglutinatingrabies virus to make it more easily phagocytosed.

In yet a further aspect, the invention includes immunoconjugates, i.e.,molecules comprising at least one binding molecule or functional variantthereof as defined herein and further comprising at least one tag, suchas inter alia a detectable moiety/agent. Also contemplated in theinvention are mixtures of immunoconjugates according to the invention ormixtures of at least one immunoconjugate according to the invention andanother molecule, such as a therapeutic agent or another bindingmolecule or immunoconjugate. In a further embodiment, theimmunoconjugates of the invention may comprise one or more tags. Thesetags can be the same or distinct from each other and can bejoined/conjugated non-covalently to the binding molecules. The tag(s)can also be joined/conjugated directly to the binding molecules throughcovalent bonding, including, but not limited to, disulfide bonding,hydrogen bonding, electrostatic bonding, recombinant fusion andconformational bonding. Alternatively, the tag(s) can bejoined/conjugated to the binding molecules by means of one or morelinking compounds. Techniques for conjugating tags to binding moleculesare well known to the skilled artisan.

The tags of the immunoconjugates of the invention may be therapeuticagents, but preferably they are detectable moieties/agents.Immunoconjugates comprising a detectable agent can be useddiagnostically to, for example, assess if a subject has been infectedwith rabies virus or monitor the development or progression of a rabiesvirus infection as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. However, they mayalso be used for other detection and/or analytical and/or diagnosticpurposes. Detectable moieties/agents include, but are not limited to,enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals, and nonradioactive paramagnetic metal ions.

The tags used to label the binding molecules for detection and/oranalytical and/or diagnostic purposes depend on the specificdetection/analysis/diagnosis techniques and/or methods used such asinter alia immunohistochemical staining of (tissue) samples, flowcytometric detection, scanning laser cytometric detection, fluorescentimmunoassays, enzyme-linked immunosorbent assays (ELISAs),radioimmunoassays (RIAs), bioassays (e.g., neutralization assays),Western blotting applications, etc. For immunohistochemical staining oftissue samples preferred labels are enzymes that catalyze production andlocal deposition of a detectable product. Enzymes typically conjugatedto binding molecules to permit their immunohistochemical visualizationare well known and include, but are not limited to,acetylcholinesterase, alkaline phosphatase, beta-galactosidase, glucoseoxidase, horseradish peroxidase, and urease. Typical substrates forproduction and deposition of visually detectable products are also wellknown to the skilled person in the art. Next to that, immunoconjugatesof the invention can be labeled using colloidal gold or they can belabeled with radioisotopes, such as ³³P, ³²P, ³⁵S, ³H, and ¹²⁵I. Bindingmolecules of the invention can be attached to radionuclides directly orindirectly via a chelating agent by methods well known in the art.

When the binding molecules of the invention are used for flow cytometricdetections, scanning laser cytometric detections, or fluorescentimmunoassays, they can usefully be labeled with fluorophores. A widevariety of fluorophores useful for fluorescently labeling the bindingmolecules of the invention are known to the skilled artisan. When thebinding molecules of the invention are used for secondary detectionusing labeled avidin, streptavidin, captavidin or neutravidin, thebinding molecules may be labeled with biotin to form suitable prostheticgroup complexes.

When the immunoconjugates of the invention are used for in vivodiagnostic use, the binding molecules can also be made detectable byconjugation to e.g., magnetic resonance imaging (MRI) contrast agents,such as gadolinium diethylenetriaminepentaacetic acid, to ultrasoundcontrast agents or to X-ray contrast agents, or by radioisotopiclabeling.

Furthermore, the binding molecules, functional variants thereof orimmunoconjugates of the invention can also be attached to solidsupports, which are particularly useful for in vitro immunoassays orpurification of rabies virus or a fragment thereof. Such solid supportsmight be porous or nonporous, planar or nonplanar and include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene supports. The human bindingmolecules can also, for example, usefully be conjugated to filtrationmedia, such as NHS-activated Sepharose or CNBr-activated Sepharose forpurposes of immunoaffinity chromatography. They can also usefully beattached to paramagnetic microspheres, typically by biotin-streptavidininteraction. The microspheres can be used for isolation of rabies virusor a fragment thereof from a sample containing rabies virus or afragment thereof. As another example, the human binding molecules of theinvention can usefully be attached to the surface of a microtiter platefor ELISA.

The binding molecules of the invention or functional variants thereofcan be fused to marker sequences, such as a peptide to facilitatepurification. Examples include, but are not limited to, thehexa-histidine tag, the hemagglutinin (HA) tag, the myc tag or the flagtag.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate. In another aspect, the human bindingmolecules of the invention may be conjugated/attached to one or moreantigens. In certain embodiments, these antigens are antigens that arerecognized by the immune system of a subject to which the bindingmolecule-antigen conjugate is administered. The antigens may beidentical but may also differ from each other. Conjugation methods forattaching the antigens and binding molecules are well known in the artand include, but are not limited to, the use of cross-linking agents.The human binding molecules will bind to rabies virus and the antigensattached to the human binding molecules will initiate a powerful T-cellattack on the conjugate which will eventually lead to the destruction ofthe rabies virus.

Next to producing immunoconjugates chemically by conjugating, directlyor indirectly via, for instance, a linker, the immunoconjugates can beproduced as fusion proteins comprising the human binding molecules ofthe invention and a suitable tag. Fusion proteins can be produced bymethods known in the art such as, e.g., recombinantly by constructingnucleic acid molecules comprising nucleotide sequences encoding thehuman binding molecules in frame with nucleotide sequences encoding thesuitable tag(s) and then expressing the nucleic acid molecules.

It is another aspect of the invention to provide a nucleic acid moleculeencoding at least a binding molecule or functional variant thereofaccording to the invention. Such nucleic acid molecules can be used asintermediates for cloning purposes, e.g., in the process of affinitymaturation described above. In a preferred embodiment, the nucleic acidmolecules are isolated or purified.

One of ordinary skill in the art will appreciate that functionalvariants of these nucleic acid molecules are also intended to be a partof the invention. Functional variants are nucleic acid sequences thatcan be directly translated, using the standard genetic code, to providean amino acid sequence identical to that translated from the parentnucleic acid molecules.

In certain embodiments, the nucleic acid molecules encode bindingmolecules comprising a CDR3 region, preferably a heavy chain CDR3region, comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ. ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

The nucleic acid molecules may encode human binding molecules comprisinga variable heavy chain comprising essentially an amino acid sequenceselected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID) NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48 and SEQ ID NO:49. In a particularly preferred embodiment, thenucleic acid molecules encode binding molecules comprising a variableheavy chain comprising essentially an amino acid sequence comprisingamino acids 1-119 of SEQ ID NO:335.

In yet another embodiment, the nucleic acid molecules encode bindingmolecules comprising a variable heavy chain comprising the amino acidsequence of SEQ ID NO:26 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:50, or they encode a variable heavy chaincomprising the amino acid sequence of SEQ ID NO:27 and a variable lightchain comprising the amino acid sequence of SEQ ID NO:51, or they encodea variable heavy chain comprising the amino acid sequence of SEQ IDNO:28 and a variable light chain comprising the amino acid sequence ofSEQ ID NO:52, or they encode a variable heavy chain comprising the aminoacid sequence of SEQ ID NO:29 and a variable light chain comprising theamino acid sequence of SEQ ID NO:53, or they encode a variable heavychain comprising the amino acid sequence of SEQ ID NO:30 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:54, or theyencode a variable heavy chain comprising the amino acid sequence of SEQID NO:31 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:55, or they encode a variable heavy chain comprising theamino acid sequence of SEQ ID NO:32 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:56, or they encode avariable heavy chain comprising the amino acid sequence of SEQ ID NO:33and a variable light chain comprising the amino acid sequence of SEQ IDNO:57, or they encode a variable heavy chain comprising the amino acidsequence of SEQ ID NO:34 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:58, or they encode a variable heavy chaincomprising the amino acid sequence of SEQ ID NO:35 and a variable lightchain comprising the amino acid sequence of SEQ ID NO:59, or they encodea variable heavy chain comprising the amino acid sequence of SEQ IDNO:36 and a variable light chain comprising the amino acid sequence ofSEQ ID NO:60, or they encode a variable heavy chain comprising the aminoacid sequence of SEQ ID NO:37 and a variable light chain comprising theamino acid sequence of SEQ ID NO:61, or they encode a variable heavychain comprising the amino acid sequence of SEQ ID NO:38 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:62, or theyencode a variable heavy chain comprising the amino acid sequence of SEQID NO:39 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:63, or they encode a variable heavy chain comprising theamino acid sequence of SEQ ID NO:40 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:64, or they encode avariable heavy chain comprising the amino acid sequence of SEQ ID NO:41and a variable light chain comprising the amino acid sequence of SEQ IDNO:65, or they encode a variable heavy chain comprising the amino acidsequence of SEQ ID NO:42 and a variable light chain comprising the aminoacid sequence of SEQ ID NO:66, or they encode a variable heavy chaincomprising the amino acid sequence of SEQ ID NO:43 and a variable lightchain comprising the amino acid sequence of SEQ ID NO:67, or they encodea variable heavy chain comprising the amino acid sequence of SEQ IDNO:44 and a variable light chain comprising the amino acid sequence ofSEQ ID NO:68, or they encode a variable heavy chain comprising the aminoacid sequence of SEQ ID NO:45 and a variable light chain comprising theamino acid sequence of SEQ ID NO:69, or they encode a variable heavychain comprising the amino acid sequence of SEQ ID NO:46 and a variablelight chain comprising the amino acid sequence of SEQ ID NO:70, or theyencode a variable heavy chain comprising the amino acid sequence of SEQID NO:47 and a variable light chain comprising the amino acid sequenceof SEQ ID NO:71, or they encode a variable heavy chain comprising theamino acid sequence of SEQ ID NO:48 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:72, or they encode avariable heavy chain comprising the amino acid sequence of SEQ ID NO:49and a variable light chain comprising the amino acid sequence of SEQ IDNO:73. In a preferred embodiment, the nucleic acid molecules encodehuman binding molecules comprising a variable heavy chain comprising theamino acid sequence comprising amino acids 1-119 of SEQ ID NO:335 and avariable light chain comprising the amino acid sequence comprising aminoacids 1-107 of SEQ ID NO:337.

In a specific embodiment of the invention, the nucleic acid moleculesencoding the variable heavy chain of the binding molecules of theinvention comprise essentially a nucleotide sequence selected from thegroup consisting of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ IDNO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96 and SEQ IDNO:97. In certain embodiments, the nucleic acid molecules encoding thevariable heavy chain of the binding molecules of the invention compriseessentially a nucleotide sequence comprising nucleotides 1-357 of SEQ IDNO:334.

In yet another specific embodiment of the invention, the nucleic acidmolecules encoding the variable light chain of the binding molecules ofthe invention comprise essentially a nucleotide sequence selected of thegroup consisting of SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ IDNO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQID NO:120 and SEQ ID NO:121. In certain embodiments, the nucleic acidmolecules encoding the variable light chain of the human bindingmolecules of the invention comprise essentially a nucleotide sequencecomprising nucleotides 1-321 of SEQ ID NO:336.

It is another aspect of the invention to provide vectors, i.e., nucleicacid constructs, comprising one or more nucleic acid molecules accordingto the invention. Vectors can be derived from plasmids such as interalia F, R1, RP1, Col, pBR322, TOL, Ti, etc.; cosmids; phages such aslambda, lambdoid, M13, Mu, P1, P22, Q_(β), T-even, T-odd, T2, T4, T7,etc.; plant viruses such as inter alia alfalfa mosaic virus, bromovirus,capillovirus, carlavirus, carmovirus, caulivirus, clostervirus,comovirus, cryptovirus, cucumovirus, dianthovirus, fabavirus, fijivirus,furovirus, geminivirus, hordeivirus, ilarvirus, luteovirus, machlovirus,marafivirus, necrovirus, nepovirus, phytorepvirus, plant rhabdovirus,potexvirus, potyvirus, sobemovirus, tenuivirus, tobamovirus, tobravirus,tomato spotted wilt virus, tombusvirus, tymovirus, etc.; or animalviruses such as inter alia adenovirus, arenaviridae, baculoviridae,birnaviridae, bunyaviridae, calciviridae, cardioviruses, coronaviridae,corticoviridae, cystoviridae, Epstein-Barr virus, enteroviruses,filoviridae, flaviviridae, Foot-and-Mouth disease virus, hepadnaviridae,hepatitis viruses, herpesviridae, immunodeficiency viruses, influenzavirus, inoviridae, iridoviridae, orthomyxoviridae, papovaviruses,paramyxoviridae, parvoviridae, picornaviridae, poliovirus,polydnaviridae, poxyiridae, reoviridae, retroviruses, rhabdoviridae,rhinoviruses, Semliki Forest virus, tetraviridae, togaviridae,toroviridae, vaccinia virus, vescular stomatitis virus, etc. Vectors canbe used for cloning and/or for expression of the human binding moleculesof the invention and might even be used for gene therapy purposes.Vectors comprising one or more nucleic acid molecules according to theinvention operably linked to one or more expression-regulating nucleicacid molecules are also covered by the invention. The choice of thevector is dependent on the recombinant procedures followed and the hostused. Introduction of vectors in host cells can be effected by interalia calcium phosphate transfection, virus infection, DEAE-dextranmediated transfection, lipofectamine transfection or electroporation.Vectors may be autonomously replicating or may replicate together withthe chromosome into which they have been integrated. In certainembodiments, the vectors contain one or more selection markers. Thechoice of the markers may depend on the host cells of choice, althoughthis is not critical to the invention as is well known to personsskilled in the art. They include, but are not limited to, kanamycin,neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene fromHerpes simplex virus (HSV-TK), and dihydrofolate reductase gene frommouse (dhfr). Vectors comprising one or more nucleic acid moleculesencoding the human binding molecules as described above operably linkedto one or more nucleic acid molecules encoding proteins or peptides thatcan be used to isolate the binding molecules are also covered by theinvention. These proteins or peptides include, but are not limited to,glutathione-S-transferase, maltose-binding protein, metal-bindingpolyhistidine, green fluorescent protein, luciferase andbeta-galactosidase.

Hosts containing one or more copies of the vectors mentioned above arean additional subject of the invention. In certain embodiments, thehosts are host cells. Host cells include, but are not limited to, cellsof mammalian, plant, insect, fungal or bacterial origin. Bacterial cellsinclude, but are not limited to, cells from Gram positive bacteria suchas several species of the genera Bacillus, Streptomyces andStaphylococcus or cells of Gram negative bacteria such as severalspecies of the genera Escherichia, such as E. coli, and Pseudomonas. Inthe group of fungal cells preferably yeast cells are used. Expression inyeast can be achieved by using yeast strains such as inter alia Pichiapastoris, Saccharomyces cerevisiae and Hansenula polymorpha.Furthermore, insect cells such as cells from Drosophila and Sf9 can beused as host cells. Besides that, the host cells can be plant cells.Transformed (transgenic) plants or plant cells are produced by knownmethods, for example, Agrobacterium-mediated gene transfer,transformation of leaf discs, protoplast transformation by polyethyleneglycol-induced DNA transfer, electroporation, sonication, microinjectionor bolistic gene transfer. Additionally, a suitable expression systemcan be a baculovirus system. Expression systems using mammalian cells,such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowesmelanoma cells, are preferred in the invention. Mammalian cells provideexpressed proteins with post-translational modifications that are mostsimilar to natural molecules of mammalian origin. Since the inventiondeals with molecules that may have to be administered to humans, acompletely human expression system would be particularly preferred.Therefore, even more preferably, the host cells are human cells.Examples of human cells are inter alia HeLa, 911, AT1080, A549, 293 andHEK293T cells. Preferred mammalian cells are human retina cells such as911 cells or the cell line deposited at the European Collection of CellCultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on29 Feb. 1996 under number 96022940 and marketed under the trademarkPER.C60 (PER.C6 is a registered trademark of Crucell Holland B.V.). Forthe purposes of this application “PER.C6” refers to cells depositedunder number 96022940 or ancestors, passages up-stream or downstream aswell as descendants from ancestors of deposited cells, as well asderivatives of any of the foregoing.

In preferred embodiments, the human producer cells comprise at least afunctional part of a nucleic acid sequence encoding an adenovirus E1region in expressible format. In even more preferred embodiments, thehost cells are derived from a human retina and immortalized with nucleicacids comprising adenoviral E1 sequences, such as the cell linedeposited at the European Collection of Cell Cultures (ECACC), CAMR,Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under number96022940 and marketed under the trademark PER.C6®. Production ofrecombinant proteins in host cells can be performed according to methodswell known in the art. The use of the cells marketed under the trademarkPER.C6® as a production platform for proteins of interest has beendescribed in WO 00/63403 the disclosure of which is incorporated hereinby reference in its entirety.

A method of producing a binding molecule or a functional variantaccording to the invention is an additional part of the invention. Themethod comprises the steps of (a) culturing a host according to theinvention under conditions conducive to the expression of the bindingmolecule or functional variant thereof, and (b) optionally, recoveringthe expressed binding molecule or functional variant thereof. Theexpressed binding molecules or functional variants thereof can berecovered from the cell free extract, but preferably they are recoveredfrom the culture medium. Methods to recover proteins, such as bindingmolecules, from cell free extracts or culture medium are well known tothe man skilled in the art. Binding molecules or functional variantsthereof as obtainable by the above described method are also a part ofthe invention.

Alternatively, next to the expression in hosts, such as host cells, thebinding molecules or functional variants thereof of the invention can beproduced synthetically by conventional peptide synthesizers or incell-free translation systems using RNA nucleic acid derived from DNAmolecules according to the invention. Binding molecule or functionalvariants thereof as obtainable by the above described syntheticproduction methods or cell-free translation systems are also a part ofthe invention.

In certain embodiments, binding molecules or functional variantsthereof, according to the invention, may be generated by transgenicnon-human mammals, such as, for instance, transgenic mice or rabbits,that express human immunoglobulin genes. In certain embodiments, thetransgenic non-human mammals have a genome comprising a human heavychain transgene and a human light chain transgene encoding all or aportion of the human binding molecules as described above. Thetransgenic non-human mammals can be immunized with a purified orenriched preparation of rabies virus or a fragment thereof. Protocolsfor immunizing non-human mammals are well established in the art. See“Using Antibodies: A Laboratory Manual,” edited by E. Harlow, D. Lane(1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and“Current Protocols in Immunology,” edited by J. E. Coligan, A. M.Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober (2001), John Wiley& Sons Inc., New York, the disclosures of which are incorporated hereinby reference.

In a further aspect, the invention provides a method of identifyingbinding molecules such as human monoclonal antibodies or fragmentsthereof according to the invention or nucleic acid molecules accordingto the invention capable of specifically binding to rabies virus andcomprises the steps of (a) contacting a collection of binding moleculeson the surface of replicable genetic packages with the rabies virus or afragment thereof under conditions conducive to binding, (b) selecting atleast once for replicable genetic packages binding to the rabies virusor the fragment thereof, and (c) separating and recovering thereplicable genetic packages binding to the rabies virus or the fragmentthereof.

The selection step may be performed in the presence of rabies virus. Therabies virus may be isolated or non-isolated, e.g., present in serumand/or blood of an infected individual. In certain embodiments, therabies virus is inactivated. Alternatively, the selection step may beperformed in the presence of a fragment of rabies virus, such as anextracellular part of the rabies virus, one or more (poly)peptidesderived from rabies virus, such as the G protein, fusion proteinscomprising these proteins or (poly)peptides, and the like. In certainembodiments, cells transfected with rabies virus G protein are used forselection procedures.

In yet a further aspect, the invention provides a method of obtaining abinding molecule or a nucleic acid molecule according to the invention,wherein the method comprises the steps of (a) performing the abovedescribed method of identifying binding molecules, such as humanmonoclonal antibodies or fragments thereof according to the invention,or nucleic acid molecules according to the invention, and (b) isolatingfrom the recovered replicable genetic packages the binding moleculeand/or the nucleic acid encoding the binding molecule. Once a newmonoclonal antibody has been established or identified with the abovementioned method of identifying binding molecules or nucleic acidmolecules encoding the binding molecules, the DNA encoding the scFv orFab can be isolated from the bacteria or replicable genetic packages andcombined with standard molecular biological techniques to makeconstructs encoding bivalent scFvs or complete human immunoglobulins ofa desired specificity (e.g., IgG, IgA or IgM). These constructs can betransfected into suitable cell lines and complete human monoclonalantibodies can be produced (see, Huls et al., 1999; Boel et al., 2000).

A replicable genetic package as used herein can be prokaryotic oreukaryotic and includes cells, spores, bacteria, viruses,(bacterio)phage and polysomes. A preferred replicable genetic package isa phage. The human binding molecules, such as, for instance, singlechain Fvs, are displayed on the replicable genetic package, i.e., theyare attached to a group or molecule located at an exterior surface ofthe replicable genetic package. The replicable genetic package is ascreenable unit comprising a human binding molecule to be screenedlinked to a nucleic acid molecule encoding the binding molecule. Thenucleic acid molecule should be replicable either in vivo (e.g., as avector) or in vitro (e.g., by PCR, transcription and translation). Invivo replication can be autonomous (as for a cell), with the assistanceof host factors (as for a virus) or with the assistance of both host andhelper virus (as for a phagemid). Replicable genetic packages displayinga collection of human binding molecules are formed by introducingnucleic acid molecules encoding exogenous binding molecules to bedisplayed into the genomes of the replicable genetic packages to formfusion proteins with endogenous proteins that are normally expressedfrom the outer surface of the replicable genetic packages. Expression ofthe fusion proteins, transport to the outer surface and assembly resultsin display of exogenous binding molecules from the outer surface of thereplicable genetic packages. In a further aspect, the invention pertainsto a human binding molecule capable of binding rabies virus or afragment thereof and being obtainable by the identification method asdescribed above.

In yet a further aspect, the invention relates to a method ofidentifying a binding molecule potentially having neutralizing activityagainst rabies virus, wherein the method comprises the steps of (a)contacting a collection of binding molecules on the surface ofreplicable genetic packages with the rabies virus under conditionsconducive to binding, (b) separating and recovering binding moleculesthat bind to the rabies virus from binding molecules that do not bind,(c) isolating at least one recovered binding molecule, (d) verifying ifthe binding molecule isolated has neutralizing activity against therabies virus, wherein the rabies virus in step a is inactivated. Theinactivated rabies virus may be purified before being inactivated.Purification may be performed by means of well-known purificationmethods suitable for viruses such as, for instance, centrifugationthrough a glycerol cushion. The inactivated rabies virus in step a maybe immobilized to a suitable material before use. Alternatively, therabies virus in step a may still be active. In another alternativeembodiment, a fragment of a rabies virus, such as a polypeptide of arabies virus such as the G protein, is used in step a. In yet anotherembodiment, cells transfected with rabies virus G protein are used forselecting binding molecule potentially having neutralizing activityagainst rabies virus. As indicated herein, when cells expressing rabiesvirus G protein were included in the selection method the number ofselected neutralizing antibodies was higher compared to selectionmethods wherein only purified rabies virus G protein and/or inactivatedrabies virus was used.

In a further embodiment, the method of identifying a binding moleculepotentially having neutralizing activity against rabies virus asdescribed above further comprises the step of separating and recovering,and optionally isolating, human binding molecules containing a variableheavy 3-30 germline gene. A person skilled in the art can identify thespecific germline gene by methods known in the art such as, forinstance, nucleotide sequencing. The step of separating and recoveringbinding molecules containing a variable heavy 3-30 germline gene can beperformed before or after step c. As indicated below the majority ofrabies virus-neutralizing human monoclonal antibodies found in theinvention comprise this specific V_(H) germline gene.

Phage display methods for identifying and obtaining (neutralizing)binding molecules, e.g., antibodies, are by now well-established methodsknown by the person skilled in the art. They are, e.g., described inU.S. Pat. No. 5,696,108; Burton and Barbas, 1994; de Kruif et al.,1995b; and “Phage Display: A Laboratory Manual,” edited by C. F. Barbas,D. R. Burton, J. K. Scott and G. J. Silverman (2001), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. All these references areherewith incorporated herein in their entirety.

For the construction of phage display libraries, collections of humanmonoclonal antibody heavy and light chain variable region genes areexpressed on the surface of bacteriophage, preferably filamentousbacteriophage, particles in, for example, single-chain Fv (scFv) or inFab format (see, de Kruif et al., 1995b). Large libraries of antibodyfragment-expressing phages typically contain more than 1.0*109 antibodyspecificities and may be assembled from the immunoglobulin V regionsexpressed in the B lymphocytes of immunized- or non-immunizedindividuals. In a specific embodiment of the invention, the phagelibrary of human binding molecules, preferably scFv phage library, isprepared from RNA isolated from cells obtained from a subject that hasbeen vaccinated against rabies or exposed to a rabies virus. RNA can beisolated from inter alia bone marrow or peripheral blood, preferablyperipheral blood lymphocytes. The subject can be an animal vaccinated orexposed to rabies virus, but is preferably a human subject which hasbeen vaccinated or has been exposed to rabies virus. In certainembodiments, the human subject has been vaccinated. A collection ofhuman binding molecules on the surface of replicable genetic packages,such as a scFv phage library, as described above is another aspect ofthe invention.

Alternatively, phage display libraries may be constructed fromimmunoglobulin variable regions that have been partially assembled invitro to introduce additional antibody diversity in the library(semi-synthetic libraries). For example, in vitro assembled variableregions contain stretches of synthetically produced, randomized orpartially randomized DNA in those regions of the molecules that areimportant for antibody specificity, e.g., CDR regions. Rabiesvirus-specific phage antibodies can be selected from the libraries byimmobilizing target antigens such as antigens from rabies virus on asolid phase and subsequently exposing the target antigens to a phagelibrary to allow binding of phages expressing antibody fragmentsspecific for the solid phase-bound antigen(s). Non-bound phages areremoved by washing and bound phages eluted from the solid phase forinfection of Escherichia coli (E. coli) bacteria and subsequentpropagation. Multiple rounds of selection and propagation are usuallyrequired to sufficiently enrich for phages binding specifically to thetarget antigen(s). If desired, before exposing the phage library totarget antigens the phage library can first be subtracted by exposingthe phage library to non-target antigens bound to a solid phase. Phagesmay also be selected for binding to complex antigens, such as complexmixtures of rabies virus proteins or (poly)peptides, host cellsexpressing one or more rabies virus proteins or (poly)peptides of rabiesvirus, or (inactivated) rabies virus itself. Antigen-specific phageantibodies can be selected from the library by incubating a solid phasewith bound thereon a preparation of inactivated rabies virus with thephage antibody library to let, for example, the scFv or Fab part of thephage bind to the proteins/polypeptides of the rabies virus preparation.After incubation and several washes to remove unbound and looselyattached phages, the phages that have bound with their scFv or Fab partto the preparation are eluted and used to infect Escherichia coli toallow amplification of the new specificity. Generally, one or moreselection rounds are required to separate the phages of interest fromthe large excess of non-binding phages. Alternatively, known proteins or(poly)peptides of the rabies virus can be expressed in host cells andthese cells can be used for selection of phage antibodies specific forthe proteins or (poly)peptides. A phage display method using these hostcells can be extended and improved by subtracting non-relevant bindersduring screening by addition of an excess of host cells comprising notarget molecules or non-target molecules that are similar, but notidentical, to the target, and thereby strongly enhance the chance offinding relevant binding molecules. (This process is referred to as theMAbstract® process. MAbstract® is a registered trademark of CrucellHolland B. V. See also, U.S. Pat. No. 6,265,150, which is incorporatedherein by reference.)

In yet a further aspect, the invention provides compositions comprisingat least one binding molecule, at least one functional variant orfragment thereof, at least one immunoconjugate according to theinvention or a combination thereof. The compositions may furthercomprise inter alia stabilizing molecules, such as albumin orpolyethylene glycol, or salts. In certain embodiments, the salts usedare salts that retain the desired biological activity of the humanbinding molecules and do not impart any undesired toxicological effects.If necessary, the human binding molecules of the invention may be coatedin or on a material to protect them from the action of acids or othernatural or non-natural conditions that may inactivate the bindingmolecules.

In yet a further aspect, the invention provides compositions comprisingat least one nucleic acid molecule as defined in the invention. Thecompositions may comprise aqueous solutions such as aqueous solutionscontaining salts (e.g., NaCl or salts as described above), detergents(e.g., SDS) and/or other suitable components.

Furthermore, the invention pertains to pharmaceutical compositionscomprising at least one n binding molecule according to the invention,at least one functional variant or fragment thereof, at least oneimmunoconjugate according to the invention, at least one compositionaccording to the invention, or combinations thereof. The pharmaceuticalcomposition of the invention further comprises at least onepharmaceutically acceptable excipient.

In certain embodiments, a pharmaceutical composition of the inventioncomprises at least one additional binding molecule, i.e., thepharmaceutical composition can be a cocktail/mixture of bindingmolecules. The pharmaceutical composition may comprise at least twobinding molecules according to the invention or at least one bindingmolecule according to the invention and at least one further anti-rabiesvirus binding molecule. The further binding molecule preferablycomprises a CDR3 region comprising the amino acid sequence of SEQ IDNO:25. The binding molecule comprising the CDR3 region comprising theamino acid sequence of SEQ ID NO:25 may be a chimeric or humanizedmonoclonal antibody or functional fragment thereof, but preferably, itis a human monoclonal antibody or functional fragment thereof. Incertain embodiments, the binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO:273. Incertain embodiments, the binding molecule comprises a light chainvariable region comprising the amino acid sequence SEQ ID NO:275. In yetanother embodiment, the binding molecule comprises a heavy and lightchain comprising the amino acid sequences of SEQ ID NO:123 and SEQ IDNO:125, respectively. The binding molecules in the pharmaceuticalcomposition should be capable of reacting with different, non-competingepitopes of the rabies virus. The epitopes may be present on the Gprotein of rabies virus and may be different, non-overlapping epitopes.The binding molecules should be of high affinity and should have a broadspecificity. In certain embodiments, they neutralize as many fixed andstreet strains of rabies virus as possible. Even more preferably, theyalso exhibit neutralizing activity towards other genotypes of theLyssavirus genus or even with other viruses of the rhabdovirus family,while exhibiting no cross-reactivity with other viruses or normalcellular proteins. In certain embodiments, the binding molecule iscapable of neutralizing escape variants of the other binding molecule inthe cocktail.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising at least two rabies virus-neutralizing binding molecules,preferably (human) binding molecules according to the invention, whereinthe binding molecules are capable of reacting with different,non-competing epitopes of the rabies virus. In certain embodiments, thepharmaceutical composition comprises a first rabies virus-neutralizingbinding molecule which is capable of reacting with an epitope located inantigenic site I of the rabies virus G protein and a second rabiesvirus-neutralizing binding molecule which is capable of reacting with anepitope located in antigenic site III of the rabies virus G protein. Theantigenic structure of the rabies glycoprotein was initially defined byLafon et al. (1983). The antigenic sites were identified using a panelof mouse mAbs and their respective mAb-resistant virus variants. Sincethen, the antigenic sites have been mapped by identification of theamino acid mutations in the glycoprotein of mab-resistant variants (see,Seif et al., 1985; Prehaud et al., 1988; and Benmansour et al., 1991).The majority of rabies-neutralizing mAbs are directed against antigenicsite II (see, Benmansour et al., 1991), which is a discontinuousconformational epitope comprising amino acids 34-42 and amino acids198-200 (see, Prehaud et al., 1988). Antigenic site III is a continuousconformational epitope at amino acids 330-338 and harbors two chargedresidues, K330 and R333, that affect viral pathogenicity (see, Seif etal., 1985; Coulon et al., 1998; and Dietzschold et al., 1983). Theconformational antigenic site I was defined by only one mAb, 509-6, andlocated at amino acid 231 (see, Benmansour et al., 1991; and Lafon etal., 1983). Antigenic site IV is known to harbor overlapping linearepitopes (see, Tordo, 1996; Bunschoten et al., 1989; Luo et al., 1997;and Ni et al., 1995). Benmansour et al. (1991) also described thepresence of minor site a located at position 342-343, which is distinctfrom antigenic site III despite its close proximity. Alignment of theCR-57 epitope with the currently known linear andconformational-neutralizing epitopes on rabies glycoprotein (FIG. 10)revealed that the CR-57 epitope is located in the same region as theconformational antigenic site I, defined by the single mAb 509-6. Basedon nucleotide and amino acid sequences of the glycoprotein of the escapeviruses of CR04-098, the epitope recognized by this antibody appears tobe located in the same region as the continuous conformational antigenicsite III.

In a preferred embodiment, the pharmaceutical composition comprises afirst rabies virus-neutralizing binding molecule comprising at least aCDR3 region, preferably heavy chain CDR3 region, comprising the aminoacid sequence of SEQ ID NO:25 and a second rabies virus-neutralizingbinding molecule comprising at least a CDR3 region, preferably heavychain CDR3 region, comprising the amino acid sequence selected from thegroup consisting of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16 and SEQ ID NO:22. More preferably, the second rabiesvirus-neutralizing binding molecule comprises at least a CDR3 region,preferably a heavy chain CDR3 region, comprising the amino acid sequenceof SEQ ID NO:14. In certain embodiments, the first rabiesvirus-neutralizing binding molecule comprises a heavy and light chaincomprising the amino acid sequences of SEQ ID NO:123 and SEQ ID NO:125,respectively, and the second rabies virus-neutralizing binding moleculecomprises a heavy and light chain comprising the amino acid sequences ofSEQ ID NO:335 and SEQ ID NO:337, respectively. In certain embodiments,the heavy and light chain of the first rabies virus-neutralizing bindingmolecule are encoded by SEQ ID NO:122 and SEQ ID NO:124, respectively,and the heavy and light chain of the second rabies virus-neutralizingbinding molecule are encoded by SEQ ID NO:334 and SEQ ID NO:336,respectively.

A pharmaceutical composition comprising two binding molecules, whereinthe pI of the binding molecules is divergent and may have a problem whenchoosing a suitable buffer which optimally stabilizes both bindingmolecules. When adjusting the pH of the buffer of the composition suchthat it increases the stability of one binding molecule, this mightdecrease the stability of the other binding molecule. Decrease ofstability or even instability of a binding molecule may lead to itsprecipitation or aggregation or to its spontaneous degradation resultingin loss of the functionality of the binding molecule. Therefore, inanother aspect, the invention provides a pharmaceutical compositioncomprising at least two binding molecules, preferably human bindingmolecules, wherein the binding molecules have isoelectric points (pI)that differ less than about 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7,0.6, 0.5, 0.4, 0.3, preferably less than (and including) 0.25 pI unitsfrom one another. The pI can be measured experimentally, e.g., by meansof isoelectric focusing, or be calculated based on the amino acidsequence of the binding molecules. In certain embodiments, the bindingmolecules are binding molecules according to the invention and thepharmaceutical composition is a pharmaceutical composition according tothe invention. In certain embodiments, the binding molecules aremonoclonal antibodies, e.g., human monoclonal antibodies such as IgG1antibodies. In certain embodiments, the binding molecules are capable ofbinding to and/or neutralizing an infectious agent, e.g., a virus, abacterium, a yeast, a fungus or a parasite. In certain embodiments, thebinding molecules are capable of binding to and/or neutralizing alyssavirus, e.g., rabies virus. In a specific embodiment, both bindingmolecules have a calculated pI that is in the range between 8.0-9.5,preferably 8.1-9.2, more preferably 8.2-8.5. In certain embodiments, thebinding molecules have the heavy chain CDR3 region of SEQ ID NO: 14 andSEQ ID NO:25, respectively.

In certain embodiments, the invention provides a cocktail of two or morehuman or other animal binding molecules, including but not limited toantibodies, wherein at least one binding molecule is derived from anantibody phage or other replicable package display technique and atleast one binding molecule is obtainable by a hybridoma technique. Whendivergent techniques are being used, the selection of binding moleculeshaving a compatible pI is also very useful in order to obtain acomposition, wherein each binding molecule is sufficiently stable forstorage, handling and subsequent use.

In certain embodiments, the binding molecules present in thepharmaceutical composition of the invention augment each other'sneutralizing activity, i.e., they act synergistically when combined. Inother words, the pharmaceutical compositions may exhibit synergisticrabies virus, and even lyssavirus, neutralizing activity. As usedherein, the term “synergistic” means that the combined effect of thebinding molecules when used in combination is greater than theiradditive effects when used individually. The ranges and ratios of thecomponents of the pharmaceutical compositions of the invention should bedetermined based on their individual potencies and tested in in vitroneutralization assays or animal models such as hamsters.

Furthermore, the pharmaceutical composition according to the inventionmay comprise at least one other therapeutic, prophylactic and/ordiagnostic agent. The further therapeutic and/or prophylactic agents maybe anti-viral agents such as ribavirin or interferon-alpha.

The binding molecules or pharmaceutical compositions of the inventioncan be tested in suitable animal model systems prior to use in humans.Such animal model systems include, but are not limited to, mice, rats,hamsters, monkeys, etc.

Typically, pharmaceutical compositions must be sterile and stable underthe conditions of manufacture and storage. The human binding molecules,variant or fragments thereof, immunoconjugates, nucleic acid moleculesor compositions of the invention can be in powder form forreconstitution in the appropriate pharmaceutically acceptable excipientbefore or at the time of delivery. In the case of sterile powders forthe preparation of sterile injectable solutions, the preferred methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Alternatively, the binding molecules, variant or fragments thereof,immunoconjugates, nucleic acid molecules or compositions of theinvention can be in solution and the appropriate pharmaceuticallyacceptable excipient can be added and/or mixed before or at the time ofdelivery to provide a unit dosage injectable form. In certainembodiments, the pharmaceutically acceptable excipient used in theinvention is suitable to high drug concentration, can maintain properfluidity and, if necessary, can delay absorption.

The choice of the optimal route of administration of the pharmaceuticalcompositions will be influenced by several factors including thephysico-chemical properties of the active molecules within thecompositions, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For instance, if necessary, the human bindingmolecules of the invention can be prepared with carriers that willprotect them against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can inter alia be used, such as ethylene vinyl acetate,poly-anhydrides, poly-glycolic acid, collagen, poly-orthoesters, andpoly-lactic acid. Furthermore, it may be necessary to coat the humanbinding molecules with, or co-administer the binding molecules with, amaterial or compound that prevents the inactivation of the human bindingmolecules. For example, the human binding molecules may be administeredto a subject in an appropriate carrier, for example, liposomes, or adiluent.

The routes of administration can generally be divided into two maincategories, oral and parenteral administration. The preferredadministration of the human binding molecules and pharmaceuticalcompositions of the invention is into and around the wound andintramuscularly in the gluteal region. Formulations of the human bindingmolecules and pharmaceutical compositions are dependent on the routes ofadministration.

In a further aspect, the binding molecules, functional variants,immunoconjugates, compositions, or pharmaceutical compositions of theinvention can be used as a medicament. Thus, a method of treatmentand/or prevention of a lyssavirus infection using the human bindingmolecules, functional variants, immunoconjugates, compositions, orpharmaceutical compositions of the invention is another part of theinvention. The lyssavirus can be a virus from any of the knowngenotypes, but is preferably rabies virus. The above-mentioned moleculesor compositions can be used in the post-exposure prophylaxis of rabies.

The molecules or compositions mentioned above may be employed inconjunction with other molecules useful in diagnosis, prophylaxis and/ortreatment of rabies virus. They can be used in vitro, ex vivo or invivo. For instance, the human binding molecules, functional variants,immunoconjugates or pharmaceutical compositions of the invention can beco-administered with a vaccine against rabies. Alternatively, thevaccine may also be administered before or after administration of themolecules or compositions of the invention. Administration of themolecules or compositions of the invention with a vaccine is suitablefor post exposure prophylaxis. Rabies vaccines include, but are notlimited to, purified chick embryo cell (PCEC) vaccine (RabAvert), humandiploid cell vaccine (HDCV; Imovax vaccine) or rabies vaccine adsorbed(RVA).

The molecules are typically formulated in the compositions andpharmaceutical compositions of the invention in a therapeutically ordiagnostically effective amount. Dosage regimens can be adjusted toprovide the optimum desired response (e.g., a therapeutic response). Asuitable dosage range may, for instance, be 0.1-100 IU/kg body weight,preferably 1.0-50 IU/kg body weight and more preferably 10-30 IU/kg bodyweight, such as 20 IU/kg body weight.

In certain embodiments, a single bolus of the binding molecules orpharmaceutical compositions of the invention are administered. Themolecules and pharmaceutical compositions according to the invention arepreferably sterile. Methods to render these molecules and compositionssterile are well known in the art. The dosing regimen of post exposureprophylaxis is administration of five doses of rabies vaccineintramuscularly in the deltoid muscle on days 0, 3, 7, 14 and 28 daysafter exposure in individuals not previously immunized against rabiesvirus. The human binding molecules or pharmaceutical compositionsaccording to the invention should be administered into and around thewounds on day 0 or otherwise as soon as possible after exposure, withthe remaining volume given intramuscularly at a site distant from thevaccine. Non-vaccinated individuals are advised to be administeredanti-rabies virus human binding molecules, but it is clear to theskilled artisan that vaccinated individuals in need of such treatmentmay also be administered anti-rabies virus human binding molecules.

In another aspect, the invention concerns the use of binding moleculesor functional variants thereof, immunoconjugates according to theinvention, nucleic acid molecules according to the invention,compositions or pharmaceutical compositions according to the inventionin the preparation of a medicament for the diagnosis, prophylaxis,treatment, or combination thereof, of a condition resulting from aninfection by a lyssavirus. The lyssavirus can be a virus from any of theknown genotypes but is preferably rabies virus. In certain embodiments,the molecules mentioned above are used in the preparation of amedicament for the post exposure prophylaxis of rabies.

Next to that, kits comprising at least one binding molecule according tothe invention, at least one functional variant thereof according to theinvention, at least one immunoconjugate according to the invention, atleast one nucleic acid molecule according to the invention, at least onecomposition according to the invention, at least one pharmaceuticalcomposition according to the invention, at least one vector according tothe invention, at least one host according to the invention or acombination thereof are also a part of the invention. Optionally, theabove described components of the kits of the invention are packed insuitable containers and labeled for diagnosis, prophylaxis and/ortreatment of the indicated conditions. The above-mentioned componentsmay be stored in unit or multi-dose containers, for example, sealedampoules, vials, bottles, syringes, and test tubes, as an aqueous,preferably sterile, solution or as a lyophilized, preferably sterile,formulation for reconstitution. The containers may be formed from avariety of materials such as glass or plastic and may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The kit may further comprise more containers comprising apharmaceutically acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, culture medium forone or more of the suitable hosts. Associated with the kits can beinstructions customarily included in commercial packages of therapeutic,prophylactic or diagnostic products, that contain information about, forexample, the indications, usage, dosage, manufacture, administration,contraindications and/or warnings concerning the use of suchtherapeutic, prophylactic or diagnostic products.

Currently, HRIG products are used for post exposure prophylaxis ofrabies. An adult dose of HRIG of 1500 IU (75 kg individual, 20 IU/kg) isonly available in a volume of 10 ml. More concentrated HRIG products arenot possible as the currently obtainable 10 ml dose contains 1-1.5 gramof total IgG. In view thereof the current HRIG products have twodrawbacks. Firstly, it is often not anatomically feasible to administerthe recommended full dose in and around the bite wounds and secondly theadministration of the current volume dose of HRIG is associated withsignificant pain. The invention gives a solution to these drawbacks asit provides a pharmaceutical composition comprising a full adult dose ina volume of approximately 2 ml or less, if desirable. Such apharmaceutical composition may comprise, for example, two bindingmolecules capable of neutralizing rabies virus, preferably CR57 andCR04-098. The pharmaceutical composition further comprises apharmaceutically acceptable excipient and has a volume of around 2 ml.More is also possible, but less desirable in view of the pain associatedwith injecting larger volumes. Less than 2 ml is also possible. Thepharmaceutical composition comprises the full adult dose (in IU)necessary for successful post exposure prophylaxis. In certainembodiments, the pharmaceutical composition is stored in a 10 ml vialsuch as, for instance, a 10 ml ready-to-use vial (type I glass) with astopper. By providing a 10 ml vial the option is given to dilute thepharmaceutical composition towards a higher volume in case an individualpresents a large wound surface area. The invention also provides a kitcomprising at least a container (such as a vial) comprising thepharmaceutical composition. The kit may further comprise a secondcontainer which holds a diluent suitable for diluting the pharmaceuticalcomposition towards a higher volume. Suitable diluents include, but arenot limited to, the pharmaceutically acceptable excipient of thepharmaceutical composition and a saline solution. Furthermore, the kitmay comprise instructions for diluting the pharmaceutical compositionand/or instructions for administering the pharmaceutical composition,whether diluted or not.

The invention further pertains to a method of detecting a rabies virusin a sample, wherein the method comprises the steps of (a) contacting asample with a diagnostically effective amount of a binding molecule, afunctional variant or an immunoconjugate according to the invention, and(b) determining whether the binding molecule, functional variant, orimmunoconjugate specifically binds to a molecule of the sample. Thesample may be a biological sample including, but not limited to blood,serum, tissue or other biological material from (potentially) infectedsubjects. The (potentially) infected subjects may be human subjects, butalso animals that are suspected as carriers of rabies virus might betested for the presence of rabies virus using the human bindingmolecules, functional variants or immunoconjugates of the invention. Thesample may first be manipulated to make it more suitable for the methodof detection. “Manipulation” means inter alia treating the samplesuspected to contain and/or containing rabies virus in such a way thatthe rabies virus will disintegrate into antigenic components such asproteins, (poly)peptides or other antigenic fragments. In certainembodiments, the binding molecules, functional variants orimmunoconjugates of the invention are contacted with the sample underconditions which allow the formation of an immunological complex betweenthe human binding molecules and rabies virus or antigenic componentsthereof that may be present in the sample. The formation of animmunological complex, if any, indicating the presence of rabies virusin the sample, is then detected and measured by suitable means. Suchmethods include, inter alia, homogeneous and heterogeneous bindingimmunoassays, such as radioimmunoassays (RIA), ELISA,immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blotanalyses.

Furthermore, the binding molecules of the invention can be used toidentify epitopes of rabies virus proteins such as the G protein. Theepitopes can be linear, but also structural and/or conformational. Inone embodiment, binding of binding molecules of the invention to aseries of overlapping peptides, such as 15-mer peptides, of a proteinfrom rabies virus such as the rabies virus G protein can be analyzed bymeans of PEPSCAN analysis (see, inter alia WO 84/03564, WO 93/09872,Slootstra et al. 1996). The binding of human binding molecules to eachpeptide can be tested in a PEPSCAN-based enzyme-linked immuno assay(ELISA). In certain embodiments, a random peptide library comprisingpeptides from rabies virus proteins can be screened for peptides capableof binding to the human binding molecules of the invention. In the aboveassays the use of rabies virus-neutralizing human binding molecules mayidentify one or more neutralizing epitopes. The peptides/epitopes foundcan be used as vaccines and for the diagnosis of rabies.

In a further aspect, the invention provides a method of screening abinding molecule or a functional variant of a binding molecule forspecific binding to a different, preferably non-overlapping epitope ofrabies virus as the epitope bound by a binding molecule or functionalvariant of the invention, wherein the method comprises the steps of (a)contacting a binding molecule or a functional variant to be screened, abinding molecule or functional variant of the invention and rabies virusor a fragment thereof (such as for instance the rabies virus G protein),(b) measure if the binding molecule or functional variant to be screenedis capable of competing for specifically binding to the rabies virus orfragment thereof with the binding molecule or functional variant of theinvention. If no competition is measured the binding molecules orfunctional variants to be screened bind to a different epitope. In aspecific embodiment of the above screening method, human bindingmolecules, or functional variants thereof, may be screened to identifyhuman binding molecules or functional variants capable of binding adifferent epitope than the epitope recognized by the binding moleculecomprising the CDR3 region comprising the amino acid sequence of SEQ IDNO:25. In certain embodiments, the epitopes are non-overlapping ornon-competing. It is clear to the skilled person that the abovescreening method can also be used to identify binding molecules orfunctional variants thereof capable of binding to the same epitope. In afurther step it may be determined if the screened binding molecules thatare not capable of competing for specifically binding to the rabiesvirus or fragment thereof have neutralizing activity. It may also bedetermined if the screened binding molecules that are capable ofcompeting for specifically binding to the rabies virus or fragmentthereof have neutralizing activity. Neutralizing anti-rabies virusbinding molecules or functional variants thereof found in the screeningmethod are another part of the invention. In the screening method“specifically binding to the same epitope” also contemplates specificbinding to substantially or essentially the same epitope as the epitopebound by the human binding molecules of the invention. The capacity toblock, or compete with, the binding of the human binding molecules ofthe invention to rabies virus typically indicates that a bindingmolecule to be screened binds to an epitope or binding site on therabies virus that structurally overlaps with the binding site on therabies virus that is immunospecifically recognized by the bindingmolecules of the invention. Alternatively, this can indicate that abinding molecule to be screened binds to an epitope or binding sitewhich is sufficiently proximal to the binding site immunospecificallyrecognized by the binding molecules of the invention to sterically orotherwise inhibit binding of the binding molecules of the invention torabies virus or a fragment thereof.

In general, competitive inhibition is measured by means of an assay,wherein an antigen composition, i.e., a composition comprising rabiesvirus or fragments (such as G proteins) thereof, is admixed withreference binding molecules and binding molecules to be screened. Incertain embodiments, the reference binding molecule may be one of thehuman binding molecules of the invention and the binding molecule to bescreened may be another human binding molecule of the invention. Incertain embodiments, the reference binding molecule may be the bindingmolecule comprising the CDR3 region comprising the amino acid sequenceof SEQ ID NO:25 and the binding molecule to be screened may be one ofthe human binding molecules of the invention. In yet another embodiment,the reference binding molecule may be one of the human binding moleculesof the invention and the binding molecule to be screened may be thebinding molecule comprising the CDR3 region comprising the amino acidsequence of SEQ ID NO:25. Usually, the binding molecules to be screenedare present in excess. Protocols based upon ELISAs are suitable for usein such simple competition studies. In certain embodiments, one maypre-mix the reference binding molecules with varying amounts of thebinding molecules to be screened (e.g., 1:10, 1:20, 1:30, 1:40, 1:50,1:60, 1:70, 1:80, 1:90 or 1:100) for a period of time prior to applyingto the antigen composition. In other embodiments, the reference bindingmolecules and varying amounts of binding molecules to be screened cansimply be admixed during exposure to the antigen composition. In anyevent, by using species or isotype secondary antibodies one will be ableto detect only the bound reference binding molecules, the binding ofwhich will be reduced by the presence of a binding molecule to bescreened that recognizes substantially the same epitope. In conducting abinding molecule competition study between a reference binding moleculeand any binding molecule to be screened (irrespective of species orisotype), one may first label the reference binding molecule with adetectable label, such as, e.g., biotin, an enzymatic, a radioactive orother label to enable subsequent identification. In these cases, onewould pre-mix or incubate the labeled reference binding molecules withthe binding molecules to be screened at various ratios (e.g., 1:10,1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100) and (optionallyafter a suitable period of time) then assay the reactivity of thelabeled reference binding molecules and compare this with a controlvalue in which no potentially competing binding molecule was included inthe incubation. The assay may again be any one of a range ofimmunological assays based upon antibody hybridization, and thereference binding molecules would be detected by means of detectingtheir label, e.g., using streptavidin in the case of biotinylatedreference binding molecules or by using a chromogenic substrate inconnection with an enzymatic label (such as3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) orby simply detecting a radioactive label. A binding molecule to bescreened that binds to the same epitope as the reference bindingmolecule will be able to effectively compete for binding and thus willsignificantly reduce reference binding molecule binding, as evidenced bya reduction in bound label. Binding molecules binding differentnon-competing epitopes will show no reduction. The reactivity of the(labeled) reference binding molecule in the absence of a completelyirrelevant binding molecule would be the control high value. The controllow value would be obtained by incubating the labeled reference bindingmolecule with unlabelled reference binding molecules of exactly the sametype, when competition would occur and reduce binding of the labeledreference binding molecule. In a test assay, a significant reduction inlabeled reference binding molecule reactivity in the presence of abinding molecule to be screened is indicative of a binding molecule thatrecognizes the same epitope, i.e., one that “cross-reacts” with thelabeled reference binding molecule. If no reduction is shown, thebinding molecule may bind a different non-competing epitope.

Binding molecules identified by these competition assays (“competitivebinding molecules”) include, but are not limited to, antibodies,antibody fragments and other binding agents that bind to an epitope orbinding site bound by the reference binding molecule as well asantibodies, antibody fragments and other binding agents that bind to anepitope or binding site sufficiently proximal to an epitope bound by thereference binding molecule for competitive binding between the bindingmolecules to be screened and the reference binding molecule to occur. Incertain embodiments, competitive binding molecules of the inventionwill, when present in excess, inhibit specific binding of a referencebinding molecule to a selected target species by at least 10%,preferably by at least 25%, more preferably by at least 50%, and mostpreferably by at least 75% to 90% or even greater. The identification ofone or more competitive binding molecules that bind to about,substantially, essentially or at the same epitope as the bindingmolecules of the invention is a straightforward technical matter. As theidentification of competitive binding molecules is determined incomparison to a reference binding molecule, it will be understood thatactually determining the epitope to which the reference binding moleculeand the competitive binding molecule bind is not in any way required inorder to identify a competitive binding molecule that binds to the sameor substantially the same epitope as the reference binding molecule.Alternatively, binding molecules binding to different non-competingepitopes identified by these competition assays may also include, butare not limited to, antibodies, antibody fragments and other bindingagents.

In another aspect, the invention provides a method of identifying abinding molecule potentially having neutralizing activity against aninfectious agent causing disease in a living being or a nucleic acidmolecule encoding a binding molecule potentially having neutralizingactivity against an infectious agent causing disease in a living being,wherein the method comprises the steps of (a) contacting a collection ofbinding molecules on the surface of replicable genetic packages with atleast a cell expressing a protein of the infectious agent causingdisease in a living being on its surface under conditions conducive tobinding, (b) separating and recovering binding molecules that bind tothe cell expressing a protein of the infectious agent causing disease ina living being on its surface from binding molecules that do not bindthe cell, (c) isolating at least one recovered binding molecule, (d)verifying if the binding molecule isolated has neutralizing activityagainst the infectious agent causing disease in a living being. The cellexpressing a protein of the infectious agent causing disease in a livingbeing on its surface can be a cell transfected with the protein. Aperson skilled in the art is aware that antigens of the infectious agentother than proteins can also be successfully used in the method. In aspecific embodiment, the cell is a PER.C6® cell. However, other(E1-immortalized) cell lines could also be used to express the proteinssuch as BHK, CHO, NSO, HEK293, or 911 cells. In certain embodiments, thebinding molecule is human. The infectious agent can be a virus, abacterium, a yeast, a fungus or a parasite. In certain embodiments, theprotein is a protein normally expressed on the surface of the infectiousagent or comprises at least a part of a protein that is surfaceaccessible. In a specific embodiment, the collection of bindingmolecules on the surface of replicable genetic packages aresubtracted/counterselected with the cells used for expressing of theprotein of the infectious agent, i.e., the cells are identical to thecells used in step a with the proviso that they do not express theprotein of the infectious agent on their surface. The cells used forsubtraction/counterselection can be untransfected cells. Alternatively,the cells can be transfected with a protein or (extracellular) partthereof that is similar and/or highly homologous in sequence orstructure with the respective protein of the infectious agent and/orthat is derived from an infectious agent of the same family or evengenus.

Another aspect of the invention pertains to a binding molecule asdefined herein having rabies virus-neutralizing activity, wherein thehuman binding molecule comprises at least a heavy chain CDR3 regioncomprising the amino acid sequence comprising SEQ ID NO:25 and furtherwherein the human binding molecule has a rabies virus-neutralizingactivity of at least 2500 IU/mg protein. More preferably, the humanbinding molecule has a rabies virus-neutralizing activity of at least2800 IU/mg protein, at least 3000 IU/mg protein, at least 3200 IU/mgprotein, at least 3400 IU/mg protein, at least 3600 IU/mg protein, atleast 3800 IU/mg protein, at least 4000 IU/mg protein, at least 4200IU/mg protein, at least 4400 IU/mg protein, at least 4600 IU/mg protein,at least 4800 IU/mg protein, at least 5000 IU/mg protein, at least 5200IU/mg protein, at least 5400 IU/mg protein. The neutralizing activity ofthe binding molecule was measured by an in vitro neutralization assay(modified RFFIT (rapid fluorescent focus inhibition test)). The assay isdescribed in detail in the example section infra.

In certain embodiments, the binding molecule comprises a variable heavychain comprising the amino acid sequence comprising SEQ ID NO:273. Incertain embodiments, the binding molecule comprises a heavy chaincomprising the amino acid sequence comprising SEQ ID NO:123. Thevariable light chain of the binding molecule may comprise the amino acidsequence comprising SEQ ID NO:275. The light chain of the bindingmolecule may comprise the amino acid sequence comprising SEQ ID NO:125.

A nucleic acid molecule encoding the binding molecules as describedabove is also a part of the invention. In certain embodiments, thenucleic acid molecule comprises the nucleotide sequence comprising SEQID NO:122. In addition, the nucleic acid molecule may also comprise thenucleotide sequence comprising SEQ ID NO:124. A vector comprising thenucleic acid molecules and a host cell comprising such a vector are alsoprovided herein. In certain embodiments, the host cell is a mammaliancell such as a human cell. Examples of cells suitable for production ofhuman binding molecules are inter alia HeLa, 911, AT1080, A549, 293 andHEK293T cells. Preferred mammalian cells are human retina cells such as911 cells or the cell line deposited at the European Collection of CellCultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on29 Feb. 1996 under number 96022940 and marketed under the trademarkPER.C6® (PER.C6 is a registered trademark of Crucell Holland B.V.). Forthe purposes of this application “PER.C6” refers to cells depositedunder number 96022940 or ancestors, passages up-stream or downstream aswell as descendants from ancestors of deposited cells, as well asderivatives of any of the foregoing.

EXAMPLES

To illustrate the invention, the following examples are provided. Theexamples are not intended to limit the scope of the invention in anyway.

Example 1 Epitope Recognition of Human Anti-Rabies Antibodies CR-57 andCR-J

To address whether the human monoclonal antibodies called CR-57 andCR-JB recognize non-overlapping, non-competing epitopes, escape virusesof the human monoclonal antibodies called CR-57 and CR-JB weregenerated. CR-57 and CR-JB were generated essentially as described (see,Jones et al., 2003), via introduction of the variable heavy and lightchain coding regions of the corresponding antibody genes into a singlehuman IgG1 expression vector named pcDNA3002(Neo). The resulting vectorspgSO57C11 and pgSOJBC11 were used for transient expression in cells fromthe cell line deposited at the European Collection of Cell Cultures(ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb.1996 under number 96022940 and marketed under the trademark PER.C6®. Thenucleotide and amino acid sequences of the heavy and light chains ofthese antibodies are shown in SEQ ID NOS:122 through 129, respectively.Serial dilutions (0.5 ml) of rabies virus strain CVS-11 (dilutionsranging from 10⁻¹ to 10⁻⁸) were incubated with a constant amount (˜4IU/ml) of antibody CR-57 or CR-JB (0.5 mil) for one hour at 37° C./5%CO₂ before addition to wells containing mouse neuroblastoma cells (MNAcells) or BSR cells (Baby Hamster Kidney-like cell line). After threedays of selection in the presence of either human monoclonal antibodyCR-57 or CR-JB, medium (1 ml) containing potential escape viruses washarvested and stored at 4° C. until further use. Subsequently, the cellswere acetone-fixed for 20 minutes at 4° C., and stained overnight at 37°C./5% CO₂ with an anti-rabies N-FITC antibody conjugate (Centocor). Thenumber of foci per well were scored by immunofluorescence and medium ofwells containing one to six foci were chosen for virus amplification.All E57 escape viruses were generated from one single focus with theexception of E57B1 (three foci). EJB escape viruses were isolated fromone focus (EJB3F), three foci (EJB2B, four foci (EJB2C), five foci(EJB2E, 2F), or six foci (EJB2D), respectively. Each escape virus wasfirst amplified on a small scale on BSR or MNA cells depending on theirgrowth characteristics. These small virus batches were then used tofurther amplify the virus on a large scale on MNA or BSR cells.Amplified virus was then titrated on MNA cells to determine the titer ofeach escape virus batch as well as the optimal dilution of the escapevirus (giving 80% to 100% infection after 24 hours) for use in a virusneutralization assay.

Modified RFFIT (rapid fluorescent focus inhibition test) assays wereperformed to examine cross-protection of E57 (the escape viruses ofCR-57) and EJB (the escape viruses of CR-JB) with CR-JB and CR-57,respectively. Therefore, CR-57 or CR-JB was diluted by serial threefolddilutions starting with a 1:5 dilution. Rabies virus (strain CVS-11) wasadded to each dilution at a concentration that gives 80% to 100%infection. Virus/IgG mix was incubated for one hour at 37° C./5% CO₂before addition to MNA cells. Twenty-four hours post-infection (at 34°C./5% CO₂) the cells were acetone-fixed for 20 minutes at 4° C., andstained for minimally three hours with an anti-rabies virus N-FITCantibody conjugate (Centocor). The wells were then analyzed for rabiesvirus infection under a fluorescence microscope to determine the 50%endpoint dilution. This is the dilution at which the virus infection isblocked by 50% in this assay. To calculate the potency, an Internat'lstandard (Rabies Immune Globulin Lot R3, Reference material from thelaboratory of Standards and Testing DMPQ/CBER/FDA) was included in eachmodified RFFIT. The 50% endpoint dilution of this standard correspondswith a potency of 2 IU/ml. The neutralizing potency of the single humanmonoclonal antibodies CR-57 and CR-JB as well as the combination ofthese antibodies were tested.

EJB viruses were no longer neutralized by CR-JB or CR-57 (see, Table 1),suggesting both antibodies bound to and induced amino acid changes insimilar regions of the rabies virus glycoprotein. E57 viruses were nolonger neutralized by CR-57, whereas 4 out of 6 E57 viruses were stillneutralized by CR-JB, although with a lower potency (see, Table 1). Amixture of the antibodies CR-57 and CR-JB (in a 1:1 IU/mg ratio) gavesimilar results as observed with the single antibodies (data not shown).

To identify possible mutations in the rabies virus glycoprotein thenucleotide sequence of the glycoprotein open reading frame (ORF) of eachof the EJB and E57 escape viruses was determined. Viral RNA of each ofthe escape viruses and CVS-11 was isolated from virus-infected MNA cellsand converted into cDNA by standard RT-PCR. Subsequently, cDNA was usedfor nucleotide sequencing of the rabies virus glycoprotein ORFs in orderto identify mutations.

Both E57 and EJB escape viruses showed mutations in the same region ofthe glycoprotein (see, FIGS. 1 and 2, respectively; see for all thesequences described in FIGS. 1 and 2 SEQ ID NOS:130 through 151). Thisindicates that both antibodies recognize overlapping epitopes. From theabove can be concluded that the combination of CR-57 and CR-JB in acocktail does not prevent the escape of neutralization-resistantvariants and is therefore not an ideal immunoglobulin preparation forrabies post exposure prophylaxis.

Example 2 Construction of a ScFv Phage Display Library using PeripheralBlood Lymphocytes of Rabies-Vaccinated Donors

From four rabies-vaccinated human subjects, 50 ml blood was drawn from avein one week after the last boost. Peripheral blood lymphocytes (PBL)were isolated from these blood samples using Ficoll cell densityfractionation. The blood serum was saved and frozen at −20° C. Thepresence of anti-rabies antibodies in the sera was tested positive usinga FACS staining on rabies virus glycoprotein transfected 293T cells.Total RNA was prepared from the PBL using organic phase separation(TRIZOL™) and subsequent ethanol precipitation. The obtained RNA wasdissolved in DEPC-treated ultrapure water and the concentration wasdetermined by OD 260 nm measurement. Thereafter, the RNA was diluted toa concentration of 100 ng/μl. Next, 1 μg of RNA was converted into cDNAas follows: To 10 μl total RNA, 13 μl DEPC-treated ultrapure water and 1μl random hexamers (500 ng/μl) were added and the obtained mixture washeated at 65° C. for five minutes and quickly cooled on wet-ice. Then, 8μl 5× First-Strand buffer, 2 μl dNTP (10 mM each), 2 μl DTT (0.1 M), 2μl Rnase-inhibitor (40 U/μl) and 2 μl Superscript™III MMLV reversetranscriptase (200 U/μl) were added to the mixture, incubated at roomtemperature for five minutes and incubated for one hour at 50° C. Thereaction was terminated by heat inactivation, i.e., by incubating themixture for 15 minutes at 75° C.

The obtained cDNA products were diluted to a final volume of 200 μl withDEPC-treated ultrapure water. The OD 260 nm of a 50 times dilutedsolution (in 10 mM Tris buffer) of the dilution of the obtained cDNAproducts gave a value of 0.1.

For each donor 5 to 10 μl of the diluted cDNA products were used astemplate for PCR amplification of the immunoglobulin gamma heavy chainfamily and kappa or lambda light chain sequences using specificoligonucleotide primers (see, Tables 2 through 7). PCR reaction mixturescontained, besides the diluted cDNA products, 25 μmol sense primer and25 μmol anti-sense primer in a final volume of 50 μl of 20 mM Tris-HCl(pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 250 μM dNTPs and 1.25 units Taqpolymerase. In a heated-lid thermal cycler having a temperature of 96°C., the mixtures obtained were quickly melted for two minutes, followedby 30 cycles of: 30 seconds at 96° C., 30 seconds at 60° C. and 60seconds at 72° C.

In a first round amplification, each of seventeen light chain variableregion sense primers (eleven for the lambda light chain (see, Table 2)and six for the kappa light chain (see, Table 3) were combined with ananti-sense primer recognizing the C-kappa called HuCk5′-ACACTCTCCCCTGTTGAAGCTCTT-3′ (see, SEQ ID NO: 152) or C-lambdaconstant region HuCλ2 5′-TGAACATTCTGTAGGGGCCACTG-3′ (see, SEQ ID NO:153)and HuCλ7 5′-AGAGCATTCTGCAGGGGCCACTG-3′ (see, SEQ ID NO:154) (the HuCλ2and HuCλ7 anti-sense primers were mixed to equimolarity before use),yielding four times 17 products of about 600 basepairs. These productswere purified on a 2% agarose gel and isolated from the gel using Qiagengel-extraction columns. One-tenth of each of the isolated products wasused in an identical PCR reaction as described above using the sameseventeen sense primers, whereby each lambda light chain sense primerwas combined with one of the three Jlambda-region-specific anti-senseprimers and each kappa light chain sense primer was combined with one ofthe five Jkappa-region-specific anti-sense primers. The primers used inthe second amplification were extended with restriction sites (see,Table 4) to enable directed cloning in the phage display vector PDV-C06(see, FIG. 3 and SEQ ID NO:155). This resulted in four times 63 productsof approximately 350 basepairs that were pooled to a total of tenfractions. This number of fractions was chosen to maintain the naturaldistribution of the different light chain families within the libraryand not to over or under represent certain families. The number ofalleles within a family was used to determine the percentage ofrepresentation within a library (see, Table 5). In the next step, 2.5 μgof pooled fraction and 100 μg PDV-C06 vector were digested with SalI andNotI and purified from gel. Thereafter, a ligation was performedovernight at 16° C. as follows. To 500 ng PDV-C06 vector 70 ng pooledfraction was added in a total volume of 50 μl ligation mix containing 50mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA and2.5 μl T4 DNA Ligase (400 U/μl). This procedure was followed for eachpooled fraction. The ligation mixes were purified by phenol/chloroform,followed by a chloroform extraction and ethanol precipitation, methodswell known to the skilled artisan. The DNA obtained was dissolved in 50μl ultrapure water and per ligation mix two times 2.5 μl aliquots wereelectroporated into 40 μl of TG1 competent E. coli bacteria according tothe manufacturer's protocol (Stratagene). Transformants were grownovernight at 37° C. in a total of 30 dishes (three dishes per pooledfraction; dimension of dish: 240 mm×240 mm) containing 2TY agarsupplemented with 50 μg/ml ampicillin and 4.5% glucose. A (sub)libraryof variable light chain regions was obtained by scraping thetransformants from the agar plates. This (sub)library was directly usedfor plasmid DNA preparation using a Qiagen™ QIAFilter MAXI prep kit.

For each donor the heavy chain immunoglobulin sequences were amplifiedfrom the same cDNA preparations in a similar two round PCR procedure andidentical reaction parameters as described above for the light chainregions with the proviso that the primers depicted in Tables 6 and 7were used. The first amplification was performed using a set of ninesense directed primers (see, Table 6; covering all families of heavychain variable regions) each combined with an IgG-specific constantregion anti-sense primer called HuCIgG 5′-GTC CAC CTT GGT GTT GCT GGGCTT-3′ (SEQ ID NO:156) yielding four times nine products of about 650basepairs. These products were purified on a 2% agarose gel and isolatedfrom the gel using Qiagen gel-extraction columns. One-tenth of each ofthe isolated products was used in an identical PCR reaction as describedabove using the same nine sense primers, whereby each heavy chain senseprimer was combined with one of the four JH-region-specific anti-senseprimers. The primers used in the second round were extended withrestriction sites (see, Table 7) to enable directed cloning in the lightchain (sub)library vector. This resulted per donor in 36 products ofapproximately 350 basepairs. These products were pooled for each donorper used (VH) sense primer into nine fractions. The products obtainedwere purified using Qiagen PCR Purification columns. Next, the fractionswere digested with SfiI and XhoI and ligated in the light chain(sub)library vector, which was cut with the same restriction enzymes,using the same ligation procedure and volumes as described above for thelight chain (sub)library. Alternatively, the fractions were digestedwith NcoI and XhoI and ligated in the light chain vector, which was cutwith the same restriction enzymes, using the same ligation procedure andvolumes as described above for the light chain (sub)library. Ligationpurification and subsequent transformation of the resulting definitivelibrary was also performed as described above for the light chain(sub)library and at this point, the ligation mixes of each donor werecombined per VH pool. The transformants were grown in 27 dishes (threedishes per pooled fraction; dimension of dish: 240 mm×240 mm) containing2TY agar supplemented with 50 μg/ml ampicillin and 4.5% glucose. Allbacteria were harvested in 2TY culture medium containing 50 μg/mlampicillin and 4.5% glucose, mixed with glycerol to 15% (v/v) and frozenin 1.5 ml aliquots at −80° C. Rescue and selection of each library wereperformed as described below.

Example 3 Selection of Phages Carrying Single Chain Fv FragmentsSpecifically Recognizing Rabies Virus Glycoprotein

Antibody fragments were selected using antibody phage display libraries,general phage display technology and MAbstract® technology, essentiallyas described in U.S. Pat. No. 6,265,150 and in WO 98/15833 (both ofwhich are incorporated by reference herein). The antibody phagelibraries used were two different semi-synthetic scFv phage libraries(JK1994 and WT2000) and the immune scFv phage libraries (RAB-03-G01 andRAB-04-G01) prepared as described in Example 2 above. The firstsemi-synthetic scFv phage library (JK1994) has been described in deKruif et al. (1995b), the second one (WT2000) was built essentially asdescribed in de Kruif et al. (1995b). Briefly, the library has asemi-synthetic format whereby variation was incorporated in the heavyand light chain V genes using degenerated oligonucleotides thatincorporate variation within CDR regions. Only VH3 heavy chain geneswere used, in combination with kappa and lambda light chain genes. CDR1and CDR3 of the heavy chain and CDR3 of the light chain were recreatedsynthetically in a PCR-based approach similar as described in de Kruifet al. (1995b). The thus created V region genes were cloned sequentiallyin scFv format in a phagemid vector and amplified to generate a phagelibrary as described before. Furthermore, the methods and helper phagesas described in WO 02/103012 (incorporated by reference herein) wereused in the invention. For identifying phage antibodies recognizingrabies virus glycoprotein phage selection experiments were performedusing whole rabies virus (rabies virus Pitman-Moore strain) inactivatedby treatment with beta-propiolactone, purified rabies virus glycoprotein(rabies virus ERA strain), and/or transfected cells expressing rabiesvirus G protein (rabies virus ERA strain).

The G protein was purified from the rabies virus ERA strain as follows.To a virus solution, 1/10 volume of 10% octyl-beta-glucopyranoside wasadded and mixed gently. Upon a 30-minute incubation at 4° C., the virussample was centrifuged (36,000 rpm, 4° C.) in a SW51 rotor. Thesupernatant was collected and dialyzed overnight at 4° C. against 0.1 MTris/EDTA. Subsequently, the glycoprotein was collected from thedialysis chamber, aliquoted, and stored at −80° C. until further use.The protein concentration was determined by OD 280 nm and the integrityof the G protein was analyzed by SDS-PAGE.

Whole inactivated rabies virus or rabies virus G protein were diluted inphosphate buffered saline (PBS), 2 to 3 ml was added to MaxiSorpNunc-Immuno Tubes (Nunc) and incubated overnight at 4° C. on a rotatingwheel. An aliquot of a phage library (500 μl, approximately 10¹³ cfu,amplified using CT helper phage (see, WO 02/103012)) was blocked inblocking buffer (2% Protifar in PBS) for one to two hours at roomtemperature. The blocked phage library was added to the immunotube(either preincubated with or without CR-57 scFv to block the epitoperecognized by CR-57), incubated for two hours at room temperature, andwashed with wash buffer (0.1% Tween-20 (Serva) in PBS) to remove unboundphages. Bound phages were then eluted from the antigen by incubation forten minutes at room temperature with 1 ml of 50 mM Glycine-HCl pH 2.2.Subsequently, the eluted phages were mixed with 0.5 ml of 1 M Tris-HClpH 7.5 to neutralize the pH. This mixture was used to infect 5 ml of anXL 1-Blue E. coli culture that had been grown at 37° C. to an OD 600 nmof approximately 0.3. The phages were allowed to infect the XL1-Bluebacteria for 30 minutes at 37° C. Then, the mixture was centrifuged forten minutes, at 3200*g at room temperature and the bacterial pellet wasresuspended in 0.5 ml 2-trypton yeast extract (2TY) medium. The obtainedbacterial suspension was divided over two 2TY agar plates supplementedwith tetracycline, ampicillin and glucose. After incubation overnight ofthe plates at 37° C., the colonies were scraped from the plates and usedto prepare an enriched phage library, essentially as described by DeKruif et al. (1995a) and WO 02/103012. Briefly, scraped bacteria wereused to inoculate 2TY medium containing ampicillin, tetracycline andglucose and grown at a temperature of 37° C. to an OD 600 nm of 0.3. CThelper phages were added and allowed to infect the bacteria after whichthe medium was changed to 2TY containing ampicillin, tetracycline andkanamycin. Incubation was continued overnight at 30° C. The next day,the bacteria were removed from the 2TY medium by centrifugation afterwhich the phages in the medium were precipitated using polyethyleneglycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml ofPBS with 1% bovine serum albumin (BSA), filter-sterilized and used forthe next round of selection.

Phage selections were also performed with rabies virus glycoproteintransfected cells. The cells used were cells from the cell linedeposited at the European Collection of Cell Cultures (ECACC), CAMR,Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996, undernumber 96022940 and marketed under the trademark PER.C60. They arehereinafter referred to as PER.C6® cells. Here, the blocked phagelibrary (2 ml) was first added to 1*107 subtractor cells (in DMEM/10%FBS) and incubated for one hour at 4° C. on a rotating wheel. Thesubtractor cells were PER.C6® cells that expressed the VesicularStomatitis Virus (VSV) glycoprotein ecto domain on their surface fusedto the rabies virus transmembrane and cytoplasmic domain. With thissubtraction step phages recognizing either VSV glycoprotein or antigensspecific for PER.C6® cells were removed from the phage library. Thephage/cell mixture was centrifuged (five minutes at 4° C. at 500×g) toremove cell-bound phages, and the supernatant was added to a new tubecontaining 3 ml of 1*107 subtractor cells. The subtraction step wasrepeated twice with the respective supernatant. Subsequently, thesubtracted phages were incubated for 1.5 hours at 4° C. on a rotatingwheel with the rabies virus glycoprotein expressing transfected cells(PER.C6® cells (3*10⁶ cells)). Before that, the transfected cells werepreincubated, either with or without CR-57 scFv, to block the epitoperecognized by CR-57. After incubation, the cells were washed five timeswith 1 ml of DMEM/10% FBS (for each wash, the cells were resuspended andtransferred to new tube), phages were eluted and processed as describedabove.

Typically, two rounds of selections were performed before isolation ofindividual phage antibodies. After the second round of selection,individual E. coli colonies were used to prepare monoclonal phageantibodies. Essentially, individual colonies were grown to log-phase in96-well plate format and infected with VCSM13 helper phages after whichphage antibody production was allowed to proceed overnight. The producedphage antibodies were PEG/NaCl-precipitated and filter-sterilized andtested in ELISA for binding to both whole inactivated rabies virus andpurified rabies virus G protein. From the selection, a large panel ofphage antibodies was obtained that demonstrated binding to both wholeinactivated rabies virus and rabies virus G protein (see, examplebelow). Two selection strategies were followed with the above-describedimmune libraries. In the first strategy 736 phage antibodies wereselected after two selection rounds using in the first and secondselection round inactivated virus or purified G protein. In the secondstrategy, 736 phage antibodies were selected after two selection roundsusing in the first selection round cell surface expressed recombinant Gprotein and in the second selection round inactivated virus or purifiedG protein. The number of unique phage antibodies obtained by the firststrategy was 97, while the second strategy yielded 70 unique ones. The97 unique phage antibodies found by means of the first strategy gaverise to 18 neutralizing antibodies and the 70 unique clones identifiedby means of the second strategy yielded 33 neutralizing antibodies. Thisclearly demonstrates that selections that included rabies virusglycoprotein transfected cells, i.e., cell surface expressed recombinantG protein, as antigen appeared to yield more neutralizing antibodiescompared to selections using only purified G protein and/or inactivatedvirus.

Example 4 Validation of the Rabies Virus Glycoprotein-SpecificSingle-Chain Phage Antibodies

Selected single-chain phage antibodies that were obtained in the screensdescribed above, were validated in ELISA for specificity, i.e., bindingto rabies virus G protein, purified as described supra. Additionally,the single-chain phage antibodies were also tested for binding to 5%FBS. For this purpose, the rabies virus G protein or 5% FBS preparationwas coated to Maxisorp™ ELISA plates. After coating, the plates wereblocked in PBS/1% Protifar for one hour at room temperature. Theselected single-chain phage antibodies were incubated for 15 minutes inan equal volume of PBS/1% Protifar to obtain blocked phage antibodies.The plates were emptied, and the blocked phage antibodies were added tothe wells. Incubation was allowed to proceed for one hour, the plateswere washed in PBS containing 0.1% Tween-20 and bound phage antibodieswere detected (using OD 492 nm measurement) using an anti-M13 antibodyconjugated to peroxidase. As a control, the procedure was performedsimultaneously using no single-chain phage antibody, a negative controlsingle chain phage antibody directed against CD8 (SCO₂-007) or apositive control single chain phage antibody directed against rabiesvirus glycoprotein (scFv SO57). As shown in Table 8, the selected phageantibodies called SC04-001, SC04-004, SC04-008, SC04-010, SC04-018,SC04-021, SC04-026, SC04-031, SC04-038, SC04-040, SC04-060, SC04-073,SC04-097, SC04-098, SC04-103, SC04-104, SC04-108, SC04-120, SC04-125,SC04-126, SC04-140, SC04-144, SC04-146, and SC04-164 displayedsignificant binding to the immobilized purified rabies virus G protein,while no binding to FBS was observed. Identical results were obtained inELISA using the whole inactivated rabies virus prepared as describedsupra (data not shown).

Example 5 Characterization of the Rabies Virus-Specific scFvs

From the selected specific single chain phage antibody (scFv) clonesplasmid DNA was obtained and nucleotide sequences were determinedaccording to standard techniques. The nucleotide sequences of the scFvs(including restriction sites for cloning) called SC04-001, SC04-004,SC04-008, SC04-010, SC04-018, SC04-021, SC04-026, SC04-031, SC04-038,SC04-040, SC04-060, SC04-073, SC04-097, SC04-098, SC04-103, SC04-104,SC04-108, SC04-120, SC04-125, SC04-126, SC04-140, SC04-144, SC04-146,and SC04-164 are shown in SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161,SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ IDNO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189,SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ IDNO:199, SEQ ID NO:201 and SEQ ID NO:203, respectively. The amino acidsequences of the scFvs called SC04-001, SC04-004, SC04-008, SC04-010,SC04-018, SC04-021, SC04-026, SC04-031, SC04-038, SC04-040, SC04-060,SC04-073, SC04-097, SC04-098, SC04-103, SC04-104, SC04-108, SC04-120,SC04-125, SC04-126, SC04-140, SC04-144, SC04-146, and SC04-164 are shownin SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ IDNO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184,SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ IDNO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202 andSEQ ID NO:204, respectively.

The VH and VL gene identity (see, I. M. Tomlinson, S. C. Williams, O.Ignatovitch, S. J. Corbett, G. Winter, “V-BASE Sequence Directory,”Cambridge United Kingdom: MRC Centre for Protein Engineering (1997)) andheavy chain CDR3 compositions of the scFvs specifically binding therabies virus G protein are depicted in Table 9.

Example 6 In vitro Neutralization of Rabies Virus by RabiesVirus-Specific scFvs (Modified RFFIT)

In order to determine whether the selected scFvs were capable ofblocking rabies virus infection, in vitro neutralization assays(modified RFFIT) were performed. The scFv preparations were diluted byserial threefold dilutions starting with a 1:5 dilution. Rabies virus(strain CVS-11) was added to each dilution at a concentration that gives80% to 100% infection. Virus/scFv mix was incubated for one hour at 37°C./5% CO₂ before addition to MNA cells. Twenty-four hours post-infection(at 34° C./5% CO₂), the cells were acetone-fixed for 20 minutes at 4°C., and stained for minimally three hours with an anti-rabies N-FITCantibody conjugate (Centocor). The cells were then analyzed for rabiesvirus infection under a fluorescence microscope to determine the 50%endpoint dilution. This is the dilution at which the virus infection isblocked by 50% in this assay (see, Example 1). Several scFvs wereidentified that showed neutralizing activity against rabies virus (see,Table 10).

Additionally, it was investigated by means of the in vitroneutralization assay (modified RFFIT) as described above, if theselected scFvs were capable of neutralizing the E57 escape viruses asprepared in Example 1 (E57A2, E57A3, E57B1, E57B2, E57B3 and E57C3).Several scFvs were identified that showed neutralizing activity againstthe E57 escape viruses (see, Tables 11A and 11B).

Example 7 Rabies Virus G Protein Competition ELISA with scFvs

To identify antibodies that bind to non-overlapping, non-competingepitopes, a rabies glycoprotein competition ELISA was performed.Nunc-Immuno™ Maxisorp F96 plates (Nunc) were coated overnight at 4° C.with a 1:1000 dilution of purified rabies virus glycoprotein (1 mg/ml;rabies virus ERA strain) in PBS (50 μl). Uncoated protein was washedaway before the wells were blocked with 100 μl PBS/1% Protifar for onehour at room temperature. Subsequently, the blocking solution wasdiscarded and 50 μl of the non-purified anti-rabies virus scFvs inPBS/1% Protifar (2× diluted) was added. Wells were washed five timeswith 100 μl of PBS/0.05% Tween-20. Then, 50 μl biotinylated anti-rabiesvirus competitor IgG, CR-57bio, was added to each well, incubated forfive minutes at room temperature, and the wells were washed five timeswith 100 μl of PBS/0.05% Tween-20. To detect the binding of CR-57bio, 50μl of a 1:2000 dilution of streptavidin-HRP antibody (Becton Dickinson)was added to the wells and incubated for one hour at room temperature.Wells were washed again as above and the ELISA was further developed byaddition of 100 μl of OPD reagens (Sigma). The reaction was stopped byadding 50 μl 1 M H₂SO₄ before measuring the OD at 492 nm.

The signal obtained with CR-57bio alone could be reduced to backgroundlevels when co-incubated with scFv SO57, i.e., the scFv form of CR-57(for nucleotide and amino acid sequence of SO57 see SEQ ID NOS:205 and206, respectively) or scFv SOJB, i.e., the scFv form of CR-JB (fornucleotide and amino acid sequence of SOJB see SEQ ID NOS:312 and 313,respectively). This indicates that the scFvs SO57 and SOJB compete withthe interaction of CR-57bio to rabies virus glycoprotein by binding tothe same epitope or to an overlapping epitope as CR-57bio, respectively.In contrast, an irrelevant scFv called SC02-007, i.e., a scFv binding toCD8, did not compete for binding. The anti-rabies virus scFvs calledSC04-004, SC04-010, SC04-024, SC04-060, SC04-073, SC04-097, SC04-098,SC04-103, SC04-104, SC04-120, SC04-125, SC04-127, SC04-140, SC04-144 andSC04-146 did also not compete with CR-57bio, indicating that these scFvsbind to a different epitope than the epitope recognized by CR-57 (see,FIG. 4).

Similar results were obtained with the following experiment. First, therabies virus antibody CR-57 was added to wells coated with rabies virusG protein. Next, the competing scFvs were added. In this set-up theanti-rabies virus scFvs were detected with anti-VSV-HRP by virtue of thepresence of a VSV-tag in the scFv amino acid sequences (see, FIG. 5).

Example 8 Construction of Fully Human Immunoglobulin Molecules (HumanMonoclonal Anti-Rabies Virus Antibodies) from the Selected Anti-RabiesVirus Single Chain Fvs

Heavy and light chain variable regions of the scFvs called SC04-001,SC04-008, SC04-018, SC04-040 and SC04-126 were PCR-amplified usingoligonucleotides to append restriction sites and/or sequences forexpression in the IgG expression vectors pSyn-CO₃—HCγ1 (see, SEQ IDNO:277) and pSyn-C04-Cλ (see, SEQ ID NO:278), respectively. The V_(H)and V_(L) genes were amplified using the oligonucleotides as shown inTable 12 and 13, respectively, and the PCR products were cloned into thevectors pSyn-C03-HCγ1 and pSyn-C04-Cλ, respectively.

Heavy and light chain variable regions of the scFvs called SC04-004,SC04-010, SC04-021, SC04-026, SC04-031, SC04-038, SC04-060, SC04-073,SC04-097, SC04-698, SC04-103, SC04-104, SC04-108, SC04-120, SC04-125,SC04-140, SC04-144, SC04-146 and SC04-164 were also PCR-amplified usingoligonucleotides to append restriction sites and/or sequences forexpression in the IgG expression vectors pSyn-C03-HCγ1 andpSyn-C05-C_(κ) (see, SEQ ID NO:279), respectively. The V_(H) and V_(L)genes were amplified using the oligonucleotides as given in Table 12 and13, respectively, and the PCR products were cloned into the vectorspSyn-C03-HCγ1 and pSyn-C05-Ck, respectively. The oligonucleotides aredesigned such that they correct any deviations from the germlinesequence that have been introduced during library construction, due tothe limited set of oligonucleotides that have been used to amplify thelarge repertoire of antibody genes. Nucleotide sequences for allconstructs were verified according to standard techniques known to theskilled artisan.

The resulting expression constructs pgG104-001C03, pgG104-008C03,pgG104-018C03, pgG104-040C03 and pgG104-126C03 encoding the anti-rabiesvirus human IgG1 heavy chains in combination with the relevantpSyn-C04-Vλ construct encoding the corresponding light chain weretransiently expressed in 293T cells and supernatants containing IgG1antibodies were obtained. The expression constructs pgG104-004C03,pgG104-010C03, pgG104-021C03, pgG104-026C03, pgG104-031C03,pgG104-038C03, pgG104-060C03, pgG104-073C03, pgG104-097C03,pgG104-098C03, pgG104-103C03, pgG104-104C03, pgG104-108C03,pgG104-120C03, pgG104-125C03, pgG104-140C03, pgG104-144C03,pgG104-146C03 and pgG104-164C03 encoding the anti-rabies virus humanIgG1 heavy chains in combination with the relevant pSyn-C15-Vκ constructencoding the corresponding light chain were transiently expressed in293T cells and supernatants containing IgG1 antibodies were obtained.

The nucleotide and amino acid sequences of the heavy and light chains ofthe antibodies called CR04-001, CR04-004, CR04-008, CR04-010, CR04-018,CR04-021, CR04-026, CR04-031, CR04-038, CR04-040, CR04-060, CR04-073,CR04-097, CR04-098, CR04-103, CR04-104, CR04-108, CR04-120, CR04-125,CR04-126, CR04-140, CR04-144, CR04-146 and CR04-164 were determinedaccording to standard techniques. Subsequently, the recombinant humanmonoclonal antibodies were purified over a protein-A column followed bya buffer exchange on a desalting column using standard purificationmethods used generally for immunoglobulins (see, for instance WO00/63403 which is incorporated by reference herein).

Additionally, for CR04-098, a single human IgG1 expression vector namedpgG104-098C10 was generated as described above for vectors pgSO57C₁₁ andpgSOJBC11 encoding CR-57 and CR-JB, respectively (see, Example 1). Thenucleotide and amino acid sequences of the heavy and light chains ofantibody CR04-098 encoded by vector pgG104-098C10 are shown in SEQ IDNOS:334 through 337, respectively. Vectors pgSO57C11 (see, Example 1)and pgG104-098C10 were used for stable expression of CR-57 and CR04-098,respectively, in cells from the cell line deposited at the EuropeanCollection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 OJG,Great Britain on 29 Feb. 1996 under number 96022940 and marketed underthe trademark PER.C6®. The stably produced CR-57 and CR04-098 have acalculated isoelectric point of 8.22 and 8.46, respectively. Theexperimentally observed isoelectric points are between 8.1-8.3 for CR-57and 9.0-9.2 for CR04-098. The recombinant human monoclonal antibodieswere purified as described above. Unless otherwise stated, for CR04-001,CR04-004, CR04-008, CR04-010, CR04-018, CR04-021, CR04-026, CR04-031,CR04-038, CR04-040, CR04-060, CR04-073, CR04-097, CR04-098, CR04-103,CR04-104, CR04-108, CR04-120, CR04-125, CR04-126, CR04-140, CR04-144,CR04-146 and CR04-164 use was made of recombinant human monoclonalantibodies transiently expressed by the two vector system as describedabove and for CR57 use was made of recombinant human monoclonal antibodytransiently expressed by the one vector system as described in Example1.

Example 9 Rabies Virus G Protein Competition ELISA with IgGs

To address whether the human monoclonal anti-rabies virus G protein IgGsbind to non-overlapping, non-competing epitopes, competition experimentsare performed. Wells with coated rabies virus G protein are incubatedwith increasing concentrations (0 to 50 μg/ml) of unlabeled anti-rabiesvirus G protein IgG for one hour at room temperature. Then, 50 μl of adifferent biotinylated anti-rabies virus IgG (1 μg/ml) is added to eachwell, incubated for five minutes at room temperature, and immediatelywashed five times with 100 μl of PBS/0.05% Tween-20. Subsequently, wellsare incubated for one hour at room temperature with 50 μl of a 1:2000dilution of streptavidin-HRP (Becton Dickinson), washed and developed asdescribed above. A decrease in signal with increasing concentration ofunlabeled IgG indicates that the two antibodies are competing with eachother and recognize the same epitope or overlapping epitopes.

Alternatively, wells coated with rabies virus G protein (ERA strain)were incubated with 50 μg/ml of unlabeled anti-rabies virus G proteinIgG for one hour at room temperature. Then, 50 μl of biotinylated CR57(0.5 to 5 μg/ml; at subsaturated levels) was added to each well. Thefurther steps were performed as described supra. The signals obtainedwere compared to the signal obtained with only biotinylated CR57 (see,FIG. 6; no competitor). From FIG. 6 can be deduced that the signal couldnot be reduced with the antibody called CR02-428 which served as anegative control. In contrast, competition with unlabeled CR57 (positivecontrol) or CR-JB reduced the signal to background levels. From FIG. 6can further be deduced that none of the anti-rabies virus G protein IgGscompeted significantly with CR-57, which is in agreement with the scFvcompetition data as described in Example 7.

In addition, competition experiments were performed on rabies virus Gprotein (ERA strain) transfected PER.C6 cells by means of flowcytometry. Transfected cells were incubated with 20 μl of unlabeledanti-rabies virus G protein IgG (50 μg/ml) for 20 minutes at 4° C. Afterwashing of the cells with PBS containing 1% BSA, 20 μl of biotinylatedCR57 (0.5 to 5 μg/ml; at subsaturated levels) were added to each well,incubated for five minutes at 4° C., and immediately washed twice with100 μl of PBS containing 1% BSA. Subsequently, wells were incubated for15 minutes at 4° C. with 20 μl of a 1:200 dilution of streptavidin-PE(Caltag), washed and developed as described above. The signal obtainedwith biotinylated CR57 could not be reduced significantly with thenegative control antibody CR02-428 (see, FIG. 7). In contrast,competition with unlabeled CR57 (positive control) or CR-JB reduced thesignal to background levels. None of the anti-rabies virus G proteinIgGs competed significantly with CR-57, with the exception of CR04-126which reduced the signal to approximately 30% (see, FIG. 7). The latterdid not compete in ELISA (see, FIG. 6). This may be caused by thedifference in the way the glycoprotein is presented to the antibody inFACS experiments compared to ELISA experiments. The binding of CR04-126could be more dependent on the conformation of the glycoprotein,resulting in the competitive effect observed with CR04-126 in theFACS-based competition assay and not in the ELISA-based competitionassay. Additionally, CR04-008 and CR04-010 reduced the signal toapproximately 50% (see, FIG. 7) in the FACS-based competition assayindicating that they might compete with CR57. For CR04-010 this washowever not confirmed by the scFv competition data or the ELISA-basedcompetition assay. For the other IgGs, the FACS data were in agreementwith the respective ELISA data of both the scFvs and the IgGs.

Example 10 Additive/Synergistic Effects of Anti-Rabies IgGs in in vitroNeutralization of Rabies Virus (Modified RFFIT)

In order to determine whether the anti-rabies virus G protein IgGs haveadditive or synergistic effects in neutralization of rabies virus,different combinations of the IgGs are tested. First, the potency (inIU/mg) of each individual antibody is determined in a modified RFFIT(see, Example 1). Then, antibody combinations are prepared based onequal amounts of IU/mg and tested in the modified RFFIT. The potenciesof each antibody combination can be determined and compared with theexpected potencies. If the potency of the antibody combination is equalto the sum of the potencies of each individual antibody present in thecombination, the antibodies have an additive effect. If the potency ofthe antibody combination is higher, the antibodies have a synergisticeffect in neutralization of rabies virus.

Alternatively, additive or synergistic effects can be determined by thefollowing experiment. First, the potency of the antibodies to be tested,e.g., CR-57 and CR04-098, is determined in a standard RFFIT (see,“Laboratory techniques in rabies,” edited by F. -X. Meslin, M. M. Kaplanand H. Koprowski (1996), 4th edition, Chapter 15, World HealthOrganization, Geneva). Then, the antibodies are mixed in a 1:1 ratiobased on IU/ml. This antibody mixture, along with the individualantibodies at the same concentration, are tested in six independentRFFIT experiments to determine the 50% neutralizing endpoint.Subsequently, the combination index (CI) is determined for the antibodymixture using the formula C1=(C1/C×1)+(C2/C×2)+(C1C2/C×1C×2) asdescribed by Chou et al. (1984). C1 and C2 are the amount (in μg) ofmonoclonal antibody 1 and monoclonal antibody 2 that lead to 50%neutralization when used in combination and C×1 and C×2 are the amount(in μg) of monoclonal antibody 1 and monoclonal antibody 2 that lead to50% neutralization when used alone. CI=1, indicates an additive effect,CI<1 indicates a synergistic effect and CI>1 indicates an antagonisticeffect of the monoclonal antibodies.

Example 11 Identification of Epitopes Recognized by Recombinant HumanAnti-Rabies Virus Antibodies by PEPSCAN-ELISA

15-mer linear and looped/cyclic peptides were synthesized from theextracellular domain of the G protein of the rabies virus strain ERA(see, SEQ ID NO:207 for the complete amino acid sequence of theglycoprotein G of the rabies virus strain ERA, the extracellular domainconsists of amino acids 20-458; the protein-id of the glycoprotein ofrabies virus strain ERA in the EMBL-database is J02293) and screenedusing credit-card format mini-PEPSCAN cards (455 peptide formats/card)as described previously (Slootstra et al., 1996; WO 93/09872). Allpeptides were acetylated at the amino terminus. In all looped peptidesposition-2 and position-14 were replaced by a cysteine(acetyl-XCXXXXXXXXXXXCX-minicard). If other cysteines besides thecysteines at position-2 and position-14 were present in a preparedpeptide, the other cysteines were replaced by an alanine. The loopedpeptides were synthesized using standard Fmoc-chemistry and deprotectedusing trifluoric acid with scavengers. Subsequently, the deprotectedpeptides were reacted on the cards with an 0.5 mM solution of1,3-bis(bromomethyl)benzene in ammonium bicarbonate (20 mM, pH7.9/acetonitril (1:1 (v/v)). The cards were gently shaken in thesolution for 30 to 60 minutes, while completely covered in the solution.Finally, the cards were washed extensively with excess of H₂O andsonicated in disrupt buffer containing 1% SDS/0.1% beta-mercaptoethanolin PBS (pH 7.2) at 70° C. for 30 minutes, followed by sonication in H₂Ofor another 45 minutes.

The human monoclonal antibodies were prepared as described above.Binding of these antibodies to each linear and looped peptide was testedin a PEPSCAN-based enzyme-linked immuno assay (ELISA). The 455-wellcredit card format polypropylene cards, containing the covalently linkedpeptides, were incubated with the antibodies (10 μg/ml; diluted inblocking solution, which contained 5% horse-serum (v/v) and 5% ovalbumin(w/v)) (4° C., overnight). After washing, the peptides were incubatedwith anti-human antibody peroxidase (dilution 1/1000) (one hour, 25°C.), and subsequently, after washing, the peroxidase substrate2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μl/ml 3% H₂O₂were added. Controls (for linear and looped) were incubated withanti-human antibody peroxidase only. After one hour, the colordevelopment was measured. The color development of the ELISA wasquantified with a CCD-camera and an image processing system. The set-upconsisted of a CCD-camera and a 55 mm lens (Sony CCD Video CameraXC-77RR, Nikon micro-nikkor 55 mm f/2.8 lens), a camera adaptor (SonyCamera adaptor DC-77RR) and the Image Processing Software packageOptimas, version 6.5 (Media Cybernetics, Silver Spring, Md. 20910,U.S.A.). Optimas ran on a Pentium II computer system.

The human anti-rabies virus G protein monoclonal antibodies were testedfor binding to the 15-mer linear and looped/cyclic peptides synthesizedas described supra. A peptide is considered to relevantly bind to anantibody when OD values are equal to or higher than two times theaverage OD-value of all peptides (per antibody). See Table 14 forresults of the binding of the human monoclonal antibodies called CR57,CRJB and CR04-010 to the linear peptides of the extracellular domain ofglycoprotein G of rabies virus strain ERA. Regions showing significantbinding to the respective antibodies are highlighted in grey.

Antibody CR57 bound to the linear peptides having an amino acid sequenceselected from the group consisting of SLKGACKLKLCGVLG (SEQ ID NO:314),LKGACKLKLCGVLGL (SEQ ID NO:315), KGACKLKLCGVLGLR (SEQ ID NO:316),GACKLKLCGVLGLRL (SEQ ID NO:317), ACKLKLCGVLGLRLM (SEQ ID NO:318),CKLKLCGVLGLRLMD (SEQ ID NO:319), KLKLCGVLGLRLMDG (SEQ ID NO:320),LKLCGVLGLRLMDGT (SEQ ID NO:321) and KLCGVLGLRLMDGTW (SEQ ID NO:322)(see, Table 14). The peptides having the amino acid sequencesGACKLKLCGVLGLRL (SEQ ID NO:317) and ACKLKLCGVLGLRLM (SEQ ID NO:318) havean OD-value that is lower than twice the average value. Neverthelessthese peptides were claimed, because they are in the near proximity of aregion of antigenic peptides recognized by antibody CR57. Binding wasmost prominent to the peptide with the amino acid sequenceKLCGVLGLRLMDGTW (SEQ ID NO:322).

Antibody CR04-010 bound to the linear peptides having an amino acidsequence selected from the group consisting of GFGKAYTIFNKTLME (SEQ IDNO:323), FGKAYTIFNKTLMEA (SEQ ID NO:324), GKAYTIFNKTLMEAD (SEQ IDNO:325), KAYTIFNKTLMEADA (SEQ ID NO:326), AYTIFNKTLMEADAH (SEQ IDNO:327), YTIFNKTLMEADAHY (SEQ ID NO:328), TIFNKTLMEADAHYK (SEQ IDNO:329), IFNKTLMEADAHYKS (SEQ ID NO:330) and FNKTLMEADAHYKSV (SEQ IDNO:331). The peptides having the amino acid sequences AYTIFNKTLMEADAH(SEQ ID NO:327) and YTFNKTLMEADAHY (SEQ ID NO:328) have an OD-value thatis lower than twice the average value. Nevertheless these peptides wereclaimed, because they are in the near proximity of a region of antigenicpeptides recognized by antibody CR04-010. Binding was most prominent tothe peptides with the amino acid sequences TIFNKTLMEADAHYK (SEQ IDNO:329), IFNKTLMEADAHYKS (SEQ ID NO:330) and FNKTLMEADAHYKSV (SEQ IDNO:331).

CRJB and the antibodies called CR04-040, CR04-098 and CR04-103 (data notshown) did not recognize a region of linear antigenic peptides.

Any of the above peptides or parts thereof represents good candidates ofa neutralizing epitope of rabies virus and could form the basis for avaccine or for raising neutralizing antibodies to treat and/or prevent arabies virus infection.

SLKGACKLKLCGVLGLRLMDGTW (SEQ ID NO:332) and GFGKAYTIFNKTLMEADAHYKSV (SEQID NO:333) are particularly interesting regions of the glycoproteinbased on their high reactivity in PEPSCAN.

From the above PEPSCAN data can further be deduced that the humanmonoclonal antibodies called CR57 and CR04-010 bind to different regionsof the rabies virus G protein indicating that they recognizenon-competing epitopes.

Example 12 Determination of Neutralizing Potency of Anti-Rabies GProtein IgGs using an in vitro Neutralization Assay (Modified RFFIT)

The neutralizing potency of each of the produced human monoclonalantibodies was determined in a modified RFFIT as described in Example 1.Sixteen IgGs neutralized rabies strain CVS-11 with a potency higher than1000 IU/mg, whereas only two IgGs had a potency lower than 2 IU/mg (see,Table 15). Eight of the sixteen antibodies outperformed transientlyproduced CR-57 with regard to potency, suggesting a higher efficiency inpost exposure prophylaxis of rabies virus than CR-57. The potency oftransiently produced CR-57 was approximately 3800 IU/mg protein (see,Tables 1 and 15), whereas stably produced CR-57 displayed a potency of5400 IU/mg protein (data not shown). Interestingly, the majority of theneutralizing human monoclonal antibodies identified contain a variableheavy 3-30 germline gene (see, Table 9).

Based on the affinity of the antibodies for rabies virus (data notshown) and 100% endpoint dilution of the antibodies in a modified RFFITassay (data not shown), a panel of six unique IgGs, i.e., CR04-010,CR04-040, CR04-098, CR04-103, CR04-104, and CR04-144, were chosen forfurther development. Within this panel, antibody CR04-098 wasparticularly interesting as it displayed the highest potency, i.e.,approximately 7300 IU/mg protein (see, Table 15). A similar potency wasalso found for stably produced CR04-098 (data not shown).

Example 13 In vitro Neutralization of E57 Escape Viruses by Anti-RabiesVirus IgGs

To further characterize the novel human monoclonal anti-rabiesantibodies the neutralizing activity of the IgGs against E57 escapeviruses was tested in a modified RFFIT as described above. The majorityof the anti-rabies virus IgGs had good neutralizing activity against allsix E57 escape viruses (see, Table 16). In contrast, CR04-008, CR04-018and CR04-126 did not neutralize 6/6, 2/6 and 3/6 E57 escape viruses,respectively. No neutralization means that no 50% endpoint was reachedat an antibody dilution of 1:100. CR04-021, CR04-108, CR04-120,CR04-125, and CR04-164 showed a significant decrease in neutralizingactivity against a number of escape viruses. This suggests that theepitope of these antibodies has been affected either directly orindirectly in the E57 escape virus glycoprotein. On the basis of theabove several anti-rabies virus IgGs may be compatible with CR-57 in ananti-rabies cocktail for post exposure prophylaxis treatment. Inparticular, the panel of six unique IgGs as identified above, i.e.,antibodies CR04-010, CR04-040, CR04-098, CR04-103, CR04-104, andCR04-144, displayed good neutralizing potency towards the E57 escapeviruses suggesting that epitope(s) recognized by these antibodieswas/were not affected by the amino acid mutations induced by CR-57.Antibody CR04-098 appeared most promising since it had a potency higherthan 3000 IU/mg for each of the escape viruses.

Example 14 Epitope Recognition of Anti-Rabies Antibodies CR-57 andCR04-098

To confirm that the human monoclonal antibodies called CR-57 andCR04-098 recognize non-overlapping, non-competing epitopes, escapeviruses of the human monoclonal antibody called CR04-098 were generatedessentially as described for escape viruses of CR57 (see, Example 1). Inshort, the number of foci per well was scored by immunofluorescence andmedium of wells containing preferably one focus were chosen for virusamplification. All E98 escape viruses were generated from one singlefocus with the exception of E98-2 (two foci) and E98-4 (four foci). Avirus was defined as an escape variant if the neutralization index was<2.5 logs. The neutralization index was determined by subtracting thenumber of infectious virus particles/ml produced in BSR cell culturesinfected with virus plus monoclonal antibody (˜4 IU/ml) from the numberof infectious virus particles/ml produced in BSR or MNA cell culturesinfected with virus alone (log focus forming units/ml virus in absenceof monoclonal antibody minus log ffu/ml virus in presence of monoclonalantibody). An index lower than 2.5 logs was considered as evidence ofescape.

To further investigate that CR04-098 binds to a differentnon-overlapping, non-competing epitope compared to CR-57, CR-57 wastested against E98 escape viruses in a modified RFFIT assay as describedabove. As shown in Table 17, CR-57 had good neutralizing activityagainst all five E98 escape viruses. Additionally, antibodies CR04-010and CR04-144 were tested for neutralizing activity against the E98escape viruses. Both antibodies did not neutralize the E98 escapeviruses (data not shown) suggesting that the epitope recognized by bothantibodies is either directly or indirectly affected by the amino acidmutation induced by antibody CR04-098. The antibodies CR04-018 andCR04-126 were tested for neutralizing activity against only one of theE98 escape viruses, i.e., E98-4. CR04-018 was capable of neutralizingthe escape virus, while CR04-126 only had a weak neutralizing potencytowards the escape virus. This suggests that the epitope recognized byCR04-018 is not affected by the mutation induced by antibody CR04-098.Additionally, the antibodies CR04-010, CR04-038, CR04-040, CR04-073,CR04-103, CR04-104, CR04-108, CR04-120, CR04-125, CR04-164 did notneutralize E98-4 suggesting that they recognize the same epitope asCR04-098 (data not shown).

To identify possible mutations in the rabies glycoprotein of each of theE98 escape viruses, the nucleotide sequence of the glycoprotein openreading frame (ORF) was determined as described before for the E57 andEJB escape viruses. All E98 escape viruses showed the mutation N to D atamino acid position 336 of the rabies glycoprotein (see, FIG. 8). Thisregion of the glycoprotein has been defined as antigenic site IIIcomprising of amino acids 330-338 (numbering without signal peptide). Incontrast, CR-57 recognized an epitope located at amino acids 226-231(numbering without signal peptide), which overlaps with antigenic siteI. In addition to the N336D mutation the E98 escape virus called E98-5showed the mutation H to Q at amino acid position 354 (codon change CATto CAG) of the rabies glycoprotein (data not shown).

Moreover, Pepscan analysis of binding of CR57 to peptides harboring amutated CR57 epitope (as observed in E57 escape viruses) did show thatinteraction of CR57 was abolished (data not shown). Strikingly, CR04-098was still capable of binding to the mutated glycoprotein (comprising theN336D mutation) expressed on PER.C6® cells, as measured by flowcytometry (data not shown), even though viruses containing this mutationwere no longer neutralized.

Furthermore, epitope mapping studies and affinity ranking studies wereperformed using surface plasmon resonance analysis using a BIAcore3000™analytical system. Purified rabies glycoprotein (ERA strain) wasimmobilized as a ligand on a research grade CM5 four-flow channel (Fc)sensor chip (Biacore AB, Sweden) using amine coupling. Ranking wasperformed at 25° C. with HBS-EP (Biacore AB, Sweden) as running buffer.50 μl of each antibody was injected at a constant flow rate of 20μl/minute. Then, running buffer was applied for 750 seconds followed byregeneration of the CM5 chip with 5 μl 2M NaOH, 5 μl 45 mM HCl and 5 μl2 mM NaOH. The resonance signals expressed as resonance units (RU) wereplotted as a function of time and the increase and decrease in RU as ameasure of association and dissociation, respectively, were determinedand used for ranking of the antibodies. The actual KD values for CR57and CR04-098 as determined by surface plasmon resonance analysis were2.4 nM and 4.5 nM, respectively. The epitope mapping studies furtherconfirmed that CR57 and CR04-098 bind to different epitopes on rabiesglycoprotein. Injection of CR57 resulted in a response of 58 RU (datanot shown). After injection of CR04-098 an additional increase inresponse level (24 RU) was obtained, suggesting that binding sites forCR04-098 were not occupied (data not shown). Similar results wereobserved when the reverse order was applied showing that each antibodyreached similar RU levels regardless of the order of injection (data notshown). These results further demonstrate that CR57 and CR04-098 canbind simultaneously and recognize different epitopes on the rabies virusglycoprotein.

Overall, the above data further confirm that the antibodies CR-57 andCR04-098 recognize distinct non-overlapping epitopes, i.e., epitopes inantigenic site I and III, respectively. The data are in good agreementwith the ELISA/FACS competition data indicating that CR-57 and CR04-098do not compete for binding to ERA G and the good neutralizing activityof antibody CR04-098 against all E57 escape viruses. On the basis ofthese results and the fact that in vitro exposure of rabies virus to thecombination of CR57 and CR04-098 (selection in the presence of 4 IU/mlof either antibody) yielded no escape viruses (data not shown), it wasconcluded that the antibodies CR-57 and CR04-098 recognizenon-overlapping, non-competing epitopes and can advantageously be usedin an anti-rabies virus antibody cocktail for post-exposure prophylaxistreatment.

Example 15 Assessment of Conservation of the Epitope Recognized by CR57and CR04-098

The minimal binding region of CR-57 (amino acids KLCGVL within SEQ IDNO:332, the region of the glycoprotein of rabies virus recognized byCR57 as determined by means of PEPSCAN and alanine scanning technology)was aligned with nucleotide sequences of 229 genotype 1 rabies virusisolates to assess the conservation of the epitope (see, Table 18). Thesample set contained human isolates, bat isolates and isolates fromcanines or from domestic animals most likely bitten by rabid canines.Frequency analysis of the amino acids at each position within theminimal binding region revealed that the critical residues constitutingthe epitope were highly conserved. The lysine at position one wasconserved in 99.6% of the isolates, while in only 1/229 isolates aconservative K>R mutation was observed. Positions two and three (L andC) were completely conserved. It is believed that the central cysteineresidue is structurally involved in the glycoprotein folding and isconserved among all lyssaviruses (see, Badrane and Tordo, 2001). Theglycine at position four was conserved in 98.7% of the isolates, whilein 3/229 isolates mutations towards charged amino acids (G>R in 1/229;G>E in 2/229) were observed. The fifth position was also conserved withthe exception of one isolate where a conservative V>I mutation wasobserved. At the sixth position, which is not a critical residue asdetermined by an alanine-replacement scan, significant heterogeneity wasobserved in the street isolates: L in 70.7%, P in 26.7% and S in 2.6% ofthe strains, respectively. Taken together, approximately 99 percent ofthe rabies viruses that can be encountered are predicted to berecognized by the CR-57 antibody.

One hundred twenty-three of these 229 virus isolates were analyzed forthe presence of mutations in both the CR-57 and CR04-098 epitope. Noneof these 123 street viruses did contain mutations in both epitopes. TheN>D mutation as observed in the E98 escape viruses was present in onlyfive virus isolates. These viruses were geographically distinct andisolated from animals in Africa (see, FIG. 9 for phylogenetic tree; thefive virus isolates, i.e., AF325483, AF325482, AF325481, AF325480 andAF325485, are indicated in bold). The phylogenetic analysis ofglycoprotein sequences revealed that rabies viruses with mutated CR57epitopes are only distantly related to rabies viruses bearing a mutatedCR04-098 epitope. Therefore, the likelihood of encountering a rabiesvirus resistant to neutralization by a cocktail of CR-57 and CR04-098 isvirtually absent.

TABLE 1 Neutralizing potency of CR-57 and CR-JB against wild-type andescape viruses. Potency Potency Potency Potency CR-57 CR-JB CR-57 CR-JBVirus (IU/mg) (IU/mg) Virus (IU/mg) (IU/mg) CVS-11 3797 605 CVS-11 3797605 E57A2 0 <0.2 EJB2B 0.004 0.6 E57A3 0 419 EJB2C <0.004 2 E57B1 0 93EJB2D <0.004 3 E57B2 0 <0.3 EJB2E <0.2 <0.3 E57B3 0 419 EJB2F <0.06 3E57C3 0 31 EJB3F <0.04 0.06

TABLE 2 Human lambda chain variable region primers (sense). Primer namePrimer nucleotide sequence SEQ ID NO HuVλ1A5′-CAGTCTGTGCTGACTCAGCCACC-3′ SEQ ID NO:208 HuVλ1B5′-CAGTCTGTGYTGACGCAGCCGCC-3′ SEQ ID NO:209 HuVλ1C5′-CAGTCTGTCGTGACGCAGCCGCC-3′ SEQ ID NO:210 HuVλ25′-CARTCTGCCCTGACTCAGCCT-3′ SEQ ID NO:211 HuVλ3A5′-TCCTATGWGCTGACTCAGCCACC-3′ SEQ ID NO:212 HuVλ3B5′-TCTTCTGAGCTGACTCAGGACCC-3′ SEQ ID NO:213 HuVλ45′-CACGTTATACTGACTCAACCGCC-3′ SEQ ID NO:214 HuVλ55′-CAGGCTGTGCTGACTCAGCCGTC-3′ SEQ ID NO:215 HuVλ65′-AATTTTATGCTGACTCAGCCCCA-3′ SEQ ID NO:216 HuVλ7/85′-CAGRCTGTGGTGACYCAGGAGCC-3′ SEQ ID NO:217 HuVλ95′-CWGCCTGTGCTGACTCAGCCMCC-3′ SEQ ID NO:218

TABLE 3 Human kappa chain variable region primers (sense). Primer namePrimer nucleotide sequence SEQ ID NO HuVκ1B5′-GACATCCAGWTGACCCAGTCTCC-3′ SEQ ID NO:219 HuVκ25′-GATGTTGTGATGACTCAGTCTCC-3′ SEQ ID NO:220 HuVκ35′-GAAATTGTGWTGACRCAGTCTCC-3′ SEQ ID NO:221 HuVκ45′-GATATTGTGATGACCCACACTCC-3′ SEQ ID NO:222 HuVκ55′-GAAACGACACTCACGCAGTCTCC-3′ SEQ ID NO:223 HuVκ65′-GAAATTGTGCTGACTCAGTCTCC-3′ SEQ ID NO:224

TABLE 4 Human kappa chain variable region primers extended with SalIrestriction sites (sense), human kappa chain J-region primers extendedwith NotI restriction sites (anti-sense), human lambda chain variableregion primers extended with SalI restriction sites (sense) and humanlambda chain J-region primers extended with NotI restriction sites(anti-sense). Primer name Primer nucleotide sequence SEQ ID NOHuVκ1B-SalI 5′-TGAGCACACAGGTCGACGGACATCCAGWTGACCCAGTCTCC-3′ SEQ IDNO:225 HuVκ2-SalI 5′-TGAGCACACAGGTCGACGGATGTTGTGATGACTCAGTCTCC-3′ SEQ IDNO:226 HuVκ3B-SalI 5′-TGAGCACACAGGTCGACGGAAATTGTGWTGACRCAGTCTCC-3′ SEQID NO:227 HuVκ4B-SalI 5′-TGAGCACACAGGTCGACGGATATTGTGATGACCCACACTCC-3′SEQ ID NO:228 HuVκ5-SalI 5′-TGAGCACACAGGTCGACGGAAACGACACTCACGCAGTCTCC-3′SEQ ID NO:229 HuVκ6-SalI 5′-TGAGCACACAGGTCGACGGAAATTGTGCTGACTCAGTCTCC-3′SEQ ID NO:230 HuJκ1-NotI5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCCACCTTGGTCCC-3′ SEQ ID NO:231HuJκ2-NotI 5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC-3′ SEQ IDNO:232 HuJκ3-NotI 5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCCACTTTGGTCCC-3′SEQ ID NO:233 HuJκ4-NotI5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCACCTTGGTCCC-3′ SEQ ID NO:234HuJκ5-NotI 5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC-3′ SEQ IDNO:235 HuVλ1A-SalI 5′-TGAGCACACAGGTCGACGCAGTCTGTGCTGACTCAGCCACC-3′ SEQID NO:236 HuVλ1B-SalI 5′-TGAGCACACAGGTCGACGCAGTCTGTGYTGACGCAGCCGCC-3′SEQ ID NO:237 HuVλ1C-SalI5′-TGAGCACACAGGTCGACGCAGTCTGTCGTGACGCAGCCGCC-3′ SEQ ID NO:238 HuVλ2-SalI5′-TGAGCACACAGGTCGACGCARTCTGCCCTGACTCAGCCT-3′ SEQ ID NO:239 HuVλ3A-SalI5′-TGAGCACACAGGTCGACGTCCTATGWGCTGACTCAGCCACC-3′ SEQ ID NO:240HuVλ3B-SalI 5′-TGAGCACACAGGTCGACGTCTTCTGAGCTGACTCAGGACCC-3′ SEQ IDNO:241 HuVλ4-SalI 5′-TGAGCACACAGGTCGACGCACGTTATACTGACTCAACCGCC-3′ SEQ IDNO:242 HuVλ5-SalI 5′-TGAGCACACAGGTCGACGCAGGCTGTGCTGACTCAGCCGTC-3′ SEQ IDNO:243 HuVλ6-SalI 5′-TGAGCACACAGGTCGACGAATTTTATGCTGACTCAGCCCCA-3′ SEQ IDNO:244 HuVλ7/8-SalI 5′-TGAGCACACAGGTCGACGCAGRCTGTGGTGACYCAGGAGCC-3′ SEQID NO:245 HuVλ9-SalI 5′-TGAGCACACAGGTCGACGCWGCCTGTGCTGACTCAGCCMCC-3′ SEQID NO:246 HuJλ1-NotI5′-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC-3′ SEQ ID NO:247HuJλ2/3-NotI 5′-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC-3′ SEQID NO:248 HuJλ4/5-NotI5′-GAGTCATTCTCGACTTGCGGCCGCACYTAAAACGGTGAGCTGGGTCCC-3′ SEQ ID NO:249

TABLE 5 Distribution of the different light chain products over the tenfractions. Light chain products Number of alleles Fraction numberalleles/fraction Vk1B/Jk1-5 19 1 and 2 9.5 Vk2/Jk1-5 9 3 9 Vk3B/Jk1-5 74 7 Vk4B/Jk1-5 1 5 5 Vk5/Jk1-5 1 Vk6/Jk1-5 3 Vλ1A/Jl1-3 5 6 5 Vλ1B/Jl1-3Vλ1C/Jl1-3 Vλ2/Jl1-3 5 7 5 Vλ3A/Jl1-3 9 8 9 Vλ3B/Jl1-3 Vλ4/Jl1-3 3 9 5Vλ5/Jl1-3 1 Vλ6/Jl1-3 1 Vλ7/8/Jl1-3 3 10 6 Vλ9/Jl1-3 3

TABLE 6 Human IgG heavy chain variable region primers (sense). Primername Primer nucleotide sequence SEQ ID NO HuVH1B/5′-CAGRTGCAGCTGGTGCARTCTGG-3′ SEQ ID NO:250 7A HuVH1C5′-SAGGTCCAGCTGGTRCAGTCTGG-3′ SEQ ID NO:251 HuVH2B5′-SAGGTGCAGCTGGTGGAGTCTGG-3′ SEQ ID NO:252 HuVH3B5′-SAGGTGCAGCTGGTGGAGTCTGG-3′ SEQ ID NO:253 HuVH3C5′-GAGGTGCAGCTGGTGGAGWCYGG-3′ SEQ ID NO:254 HuVH4B5′-CAGGTGCAGCTACAGCAGTGGGG-3′ SEQ ID NO:255 HuVH4C5′-CAGSTGCAGCTGCAGGAGTCSGG-3′ SEQ ID NO:256 HuVH5B5′-GARGTGCAGCTGGTGCAGTCTGG-3′ SEQ ID NO:257 HuVH6A5′-CAGGTACAGCTGCAGCAGTCAGG-3′ SEQ ID NO:258

TABLE 7 Human IgG heavy chain variable region primers extended withSfiI/NcoI restriction sites (sense) and human IgG heavy chain J-regionprimers extended with XhoI/BstEII restriction sites (anti-sense). Primername Primer nucleotide sequence SEQ ID NO HuVH1B/7A-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGRTGCAGCTGGTGCARTCTGG-3′ SEQ IDNO:259 HuVH1C-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSAGGTCCAGCTGGTRCAGTCTGG-3′ SEQ IDNO:260 HuVH2B-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGRTCACCTTGAAGGAGTCTGG-3′ SEQ IDNO:261 HuVH3B-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCSAGGTGCAGCTGGTGGAGTCTGG-3′ SEQ IDNO:262 HuVH3C-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGWCYGG-3′ SEQ IDNO:263 HuVH4B-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTACAGCAGTGGGG-3′ SEQ IDNO:264 HuVH4C-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGSTGCAGCTGCAGGAGTCSGG-3′ SEQ IDNO:265 HuVH5B-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGARGTGCAGCTGGTGCAGTCTGG-3′ SEQ IDNO:266 HuVH6A-SfiI5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG-3′ SEQ IDNO:267 HuJH1/2-XhoI 5′-GAGTCATTCTCGACTCGAGACGGTGACCAGGGTGCC-3′ SEQ IDNO:268 HuJH3-XhoI 5′-GAGTCATTCTCGACTCGAGACGGTGACCATTGTCCC-3′ SEQ IDNO:269 HuJH4/5-XhoI 5′-GAGTCATTCTCGACTCGAGACGGTGACCAGGGTTCC-3′ SEQ IDNO:270 HuJH6-XhoI 5′-GAGTCATTCTCGACTCGAGACGGTGACCGTGGTCCC-3′ SEQ IDNO:271

TABLE 8 Binding of single-chain (scFv) phage antibodies to rabies virusG protein (ERA strain) and to FBS as measured by ELISA. Rabies FBS Namephage antibody virus G protein (OD 492 nm) (OD 492 nm) SC04-001 0.8280.053 SC04-004 0.550 0.054 SC04-008 0.582 0.058 SC04-010 0.915 0.043SC04-018 0.247 0.052 SC04-021 0.278 0.052 SC04-026 0.212 0.054 SC04-0310.721 0.065 SC04-038 0.653 0.061 SC04-040 0.740 0.053 SC04-060 0.9230.056 SC04-073 0.657 0.054 SC04-097 0.835 0.056 SC04-098 0.798 0.060SC04-103 0.606 0.059 SC04-104 0.566 0.063 SC04-108 0.363 0.052 SC04-1200.571 0.052 SC04-125 0.735 0.049 SC04-126 0.232 0.051 SC04-140 0.8650.057 SC04-144 0.775 0.054 SC04-146 0.484 0.057 SC04-164 0.547 0.057control (SO57) 0.650 0.055 control (02-007) 0.063 0.052

TABLE 9 Data of the single-chain Fvs capable of binding rabies virus Gprotein. SEQ ID NO SEQ ID NO of nucl. of amino acid Name scFv (libr.)sequence sequence HCDR3 (SEQ ID NO:) V_(H)-locus V_(L)-locus sc04-001(JK1994) 157 158 GLYGELFDY 3-20 (DP32) Vl3 (3l-V2-13) (SEQ ID NO:1)sc04-004 (WT2000) 159 160 DYLYPTTDFDY 3-23 (DP47) VkI (O12/O2-DPK9) (SEQID NO:2) sc04-008 (RAB-03-G01) 161 162 MGFTGTYFDY 2-70 (DP28) Vl3(3h-V2-14) (SEQ ID NO:3) sc04-010 (RAB-03-G01) 163 164 DGLDLTGTIQPFGY3-30 (DP49) VkI (L11-DPK3) (SEQ ID NO:4) sc04-018 (RAB-03-G01) 165 166VSVTTGAFNI 4-04 (DP70) Vl1 (1c-V1-16) (SEQ ID NO:5) sc04-021(RAB-03-G01) 167 168 GSVLGDAFDI 3-30 (DP49) VkI (L8) (SEQ ID NO:6)sc04-026 (RAB-03-G01) 169 170 TSNWNYLDRFDP 5-51 (DP73) VkII(A19/03-DPK15) (SEQ ID NO:7) sc04-031 (RAB-03-G01) 171 172 GSVLGDAFDI3-30 (DP49) VkI (L5-DPK5) (SEQ ID NO:8) sc04-038 (RAB-03-G01) 173 174GSVLGDAFDI 3-30 (DP49) VkI (L5-DPK5) (SEQ ID NO:9) sc04-040 (RAB-03-G01)175 176 GSKVGDFDY 3-30 (DP49) Vl3 (3h-V2-14) (SEQ ID NO:10) sc04-060(RAB-04-G01) 177 178 EKEKYSDRSGYSYYYYYMDV 4-59 (DP71) VkI (O12/O2-DPK9)(SEQ ID NO:11) sc04-073 (RAB-04-G01) 179 180 DGLDLTGTIQPFGY 3-30 (DP49)VkI (L12) (SEQ ID NO:12) sc04-097 (RAB-04-G01) 181 182 TASNLGRGGMDV 3-23(DP47) VkI (L8) (SEQ ID NO:13) sc04-098 (RAB-04-G01) 183 184 VAVAGTHFDY3-30 (DP49) VkI (A30) (SEQ ID NO:14) sc04-103 (RAB-04-G01) 185 186VAVAGESFDS 3-30 (DP49) VkI (L5-DPK5) (SEQ ID NO:15) sc04-104(RAB-04-G01) 187 188 IVVVTALDAFDI 3-30 (DP49) VkI (L12) (SEQ ID NO:16)sc04-108 (RAB-04-G01) 189 190 FMIVADDAFDI 3-30 (DP49) VkI (L1) (SEQ IDNO:17) sc04-120 (RAB-04-G01) 191 192 GGKTGEFDY 3-30 (DP49) VkI (L8) (SEQID NO:18) sc04-125 (RAB-04-G01) 193 194 IATAGTGFDY 3-30 (DP49) VkI (L8)(SEQ ID NO:19) sc04-126 (RAB-04-G01) 195 196 MGFTGTYFDY 2-70 (DP28) Vl3(3h-V2-14) (SEQ ID NO:20) sc04-140 (RAB-04-G01) 197 198 VTNPGDAFDI 3-30(DP49) VkI (L4/18a) (SEQ ID NO:21) sc04-144 (RAB-04-G01) 199 200GGKTGEFDY 3-30 (DP49) VkI (L8) (SEQ ID NO:22) sc04-146 (RAB-04-G01) 201202 GGKTGEFDY 3-30 (DP49) VkIII (L2-DPK21) (SEQ ID NO:23) sc04-164(RAB-04-G01) 203 204 GSVLGDAFDI 3-30 (DP49) VkI (L19-DPK6) (SEQ IDNO:24) SO57 205 206 ENLDNSGTYYYFSGWFDP 1-69 (DP10) Vl2 (2e-V1-3) (SEQ IDNO:25) SOJB 312 313 RQHISSFPWFDS 2-05 Vl3 (3h-V2-14) (SEQ ID NO:276)

TABLE 10 Data of assay for rabies virus-neutralizing activity of scFvs.50% endpoint dilution Potency Name scFv 50% endpoint dilution WHOstandard (2 IU/ml) (IU/ml) SC04-001 270 405 1.3 SC04-004 3645 405 18SC04-008 >10935 405 >54 SC04-010 810 405 4 SC04-018 15 405 0.1 SC04-021270 405 1.3 SC04-026 45 270 0.3 SC04-031 90 270 0.7 SC04-038 270 270 2SC04-040 45 270 0.3 SC04-060 30 270 0.2 SC04-073 405 270 3 SC04-097 30270 0.2 SC04-098 1215 270 9 SC04-103 45 270 0.3 SC04-104 135 270 1SC04-108 135 270 1 SC04-120 810 270 6 SC04-125 405 270 3 SC04-126 10 2700.1 SC04-140 135 270 1 SC04-144 810 270 6 SC04-146 405 270 3 SC04-164 45270 0.3

TABLE 11A Data of assay for measuring neutralizing activity of scFvs forE57 escape viruses E57A2, E57A3 and E57B1. E57A2 E57A3 E57B1 Name scFv1* 2* 3* 1* 2* 3* 1* 2* 3* SC04-001 10 90 0.2 10 90 0.2 30 45 1.3SC04-004 810 90 18.0 1215 90 27.0 810 45 36.0 SC04-008 10 90 0.2 15 900.3 270 45 12.0 SC04-010 270 90 6.0 270 90 6.0 270 45 12.0 SC04-018 5 900.1 15 90 0.3 15 45 0.7 SC04-021 10 90 0.2 30 90 0.7 10 90 0.2 SC04-026<5 90 0.0 <5 45 0.0 <5 90 0.0 SC04-031 10 90 0.2 30 90 0.7 10 90 0.2SC04-038 90 90 2.0 90 90 2.0 45 90 1.0 SC04-040 15 90 0.3 5 90 0.1 5 900.1 SC04-060 5 90 0.1 5 90 0.1 <5 90 0.0 SC04-073 135 90 3.0 90 30 6.030 30 2.0 SC04-097 <5 90 0.0 <5 90 0.0 <5 90 0.0 SC04-098 810 90 18.0270 30 18.0 270 30 18.0 SC04-103 <5 90 0.0 10 90 0.2 5 90 0.1 SC04-10490 90 2.0 30 30 2.0 30 30 2.0 SC04-108 15 90 0.3 <5 90 0.0 <5 90 0.0SC04-120 45 90 1.0 30 30 2.0 10 30 0.7 SC04-125 135 90 3.0 135 30 9.0 9030 6.0 SC04-126 <5 90 0.0 <5 45 0.0 <5 90 0.0 SC04-140 30 45 1.3 90 306.0 45 90 1.0 SC04-144 270 45 12.0 270 30 18.0 135 90 3.0 SC04-146 90 454.0 90 30 6.0 90 90 2.0 SC04-164 15 45 0.7 30 30 2.0 15 90 0.3 1* is 50%endpoint dilution 2* is 50% endpoint dilution WHO standard (2 IU/ml) 3*is Potency (IU/ml)

TABLE 11B Data of assay for measuring neutralizing activity of scFvs forE57 escape viruses E57B2, E57B3 and E57C3. E57B2 E57B3 E57C3 Name scFv1* 2* 3* 1* 2* 3* 1* 2* 3* SC04-001 30 45 1.3 90 270 0.7 5 90 0.1SC04-004 5 45 0.2 2430 270 18.0 270 90 6.0 SC04-008 5 45 0.2 45 270 0.310 90 0.2 SC04-010 45 45 2.0 405 270 3.0 270 90 6.0 SC04-018 15 45 0.715 270 0.1 30 90 0.7 SC04-021 10 90 0.2 30 270 0.2 10 90 0.2 SC04-026 <545 0.0 <5 45 0.0 <5 30 0.0 SC04-031 10 90 0.2 30 270 0.2 30 90 0.7SC04-038 30 90 0.7 90 270 0.7 90 90 2.0 SC04-040 5 90 0.1 15 135 0.2 1090 0.2 SC04-060 <5 90 0.0 10 135 0.1 5 90 0.1 SC04-073 30 90 0.7 90 2700.7 90 90 2.0 SC04-097 <5 90 0.0 <5 135 0.0 <5 90 0.0 SC04-098 90 90 2.0810 270 6.0 270 90 6.0 SC04-103 <5 90 0.0 10 135 0.1 10 90 0.2 SC04-10445 90 1.0 45 270 0.3 90 90 2.0 SC04-108 10 90 0.2 <5 135 0.0 15 90 0.3SC04-120 15 90 0.3 45 270 0.3 30 90 0.7 SC04-125 90 90 2.0 270 270 2.0270 90 6.0 SC04-126 <5 45 0.0 <5 45 0.0 <5 30 0.0 SC04-140 30 90 0.7 9090 2.0 270 90 6.0 SC04-144 90 90 2.0 270 90 6.0 405 90 9.0 SC04-146 3090 0.7 90 90 2.0 90 90 2.0 SC04-164 15 90 0.3 15 90 0.3 30 90 0.7 1* is50% endpoint dilution 2* is 50% endpoint dilution WHO standard (2 IU/ml)3* is Potency (IU/ml)

TABLE 12 Oligonucleotides used for PCR amplification of V_(H) genes. SEQID Name and nucleotide sequence V_(H) gene NO: 5H-B:acctgtcttgaattctccatggccgaggtg SC04-001 280 cagctggtggagtctg 5H-C:acctgtcttgaattctccatggcccaggtg SC04-021 281 cagctggtggagtctgg SC04-031SC04-125 SC04-164 5H-C-long: acctgtcttgaattctccatggccc SC04-010 282aggtgcagctggtggagtctgggg SC04-038 SC04-040 SC04-073 SC04-098 SC04-103SC04-104 SC04-108 SC04-120 SC04-140 SC04-144 SC04-146 5H-F:acctgtcttgaattctccatggcccaggtg SC04-018 283 cagctgcaggagtccggcccSC04-060 5H-H: acctgtcttgaattctccatggccgaggtg SC04-026 284cagctggtgcagtctgg 5H-I: acctgtcttgaattctccatggccgaggtg SC04-004 285cagctgctggagtctgg SC04-097 5H-M: acctgtcttgaattctccatggcccaggtg SC04-008286 accttgaaggagtctgg SC04-126 sy3H-A: gcccttggtgctagcgctggagacggtcSC04-001 287 accagggtgccctggcccc SC04-004 SC04-008 SC04-010 SC04-026SC04-040 SC04-073 SC04-098 SC04-120 SC04-125 SC04-126 SC04-144 SC04-146sy3H-C: gcccttggtgctagcgctggagacggtc SC04-097 288 acggtggtgccctggccccsy3H-C-long: gcccttggtgctagcgctggaga SC04-060 289cggtcacggtggtgcccttgccccagacgtc sy3H-D: gcccttggtgctagcgctggacacggtcSC04-018 290 accatggtgccctggcccc SC04-021 SC04-031 SC04-038 SC04-104SC04-108 SC04-140 SC04-164 sy3H-E: gcccttggtgctagcgctggacacggtc SC04-103291 accagggtgccccggcccc

TABLE 13 Oligonucleotides used for PCR amplification of V_(L) genes. SEQID Name and nucleotide sequence V_(L) gene NO: 3L-B:ttttccttagcggccgcgactcacctagga SC04-001 292 cggtcagcttggtc 5K-B:acctgtctcgagttttccatggctgacatc SC04-031 293 cagatgacccagtc SC04-060SC04-073 SC04-098 SC04-103 SC04-104 SC04-108 SC04-164 5K-C:acctgtctcgagttttccatggctgacatc SC04-004 294 cagatgacccagtctccatcctccc5K-G: acctgtctcgagttttccatggctgacatc SC04-026 295 gtgatgacccagtctcc5K-K: acctgtctcgagttttccatggctgccatc SC04-010 296 cagatgacccagtctcc5K-M: acctgtctcgagttttccatggctgacatc SC04-021 297 cagctgacccagtcSC04-097 SC04-120 SC04-125 SC04-144 5K-N: acctgtctcgagttttccatggctgacatcSC04-038 298 cagatgactcagtc 5K-O: acctgtctcgagttttccatggctgccatcSC04-140 299 cagctgacccagtc 5K-Q: acctgtctcgagttttccatggctgagatcSC04-146 300 gtgatgactcagtc 5L-E: acctgtctcgagttttccatggcttcctacSC04-008 301 gtgctgactcagccg 5L-F: acctgtctcgagttttccatggctcagtccSC04-018 302 gtgctgactcagcc 5L-G: acctgtctcgagttttccatggcttcctacSC04-040 303 gtgctgactcagcc SC04-126 sy3K-F:gctgggggcggccacggtccgcttgatc SC04-004 304 tccaccttggtccc SC04-010SC04-021 SC04-031 SC04-098 SC04-104 SC04-125 SC04-140 SC04-144 SC04-164sy3K-I: gctgggggcggccacggtccgcttgatc SC04-038 305 tccagccgtgtcccSC04-097 SC04-103 SC04-108 SC04-146 sy3K-J: gctgggggcggccacggtccgcttgatcSC04-026 306 tccagcttggtccc SC04-060 SC04-073 sy3K-K:gctgggggcggccacggtccgcttgatg SC04-120 307 tccaccttggtccc sy3L-A:ccagcacggtaagcttcagcacggtcac SC04-018 308 cttggtgccagttcc SC04-126sy3L-C: ccagcacggtaagcttcagcacggtcag SC04-040 309 cttggtgcctccgccsy3L-D: ccagcacggtaagcttcaacacggtcag SC04-008 310 ctgggtccc sy5L-A:acctgtctcgagttttccatggcttcct SC04-001 311 ccgagctgacccaggaccctgctg

TABLE 14 Binding of the human monoclonal antibodies CR57, CRJB andCR04-010 (10 μg/ml) to linear peptides of the extracellular domain ofglycoprotein G of rabies virus strain ERA. Amino acid sequence of linearpeptide CR57 CRJB CR04-010 SEQ ID NO: KFPIYTWDKLGPWS 71 97 1 338FPIYTILDKLGPWSP 42 105 39 339 PIYTILDKLGPWSPI 36 89 87 340IYTILDKLGPWSPID 44 97 104 341 YTILDKLGPWSPIDI 48 114 91 342TILDKLGPWSPIIMH 76 96 88 343 ILDKLGPWSPIDIHH 54 104 69 344LDKLGPWSPIDIHHL 55 99 107 345 DKLGPWSPIDIHHLS 62 103 93 346KLGPWSPIDIHHLSC 72 105 45 347 LGPWSPIDIHHLSCP 69 112 19 348GPWSPIDIHHLSCPN 68 114 33 349 PWSPIDIHHLSCPNN 62 104 47 350WSPIDIHHLSCPNNL 80 106 11 351 SPIDIHHLSCPNNLV 74 85 1 352PIDLHHLSCPNNLVV 46 93 90 353 IDIHHLSCPNNLVVE 69 102 55 354DIHHLSCPNNLVVED 38 96 78 355 IHHLSCPNNLVVEDE 37 85 113 356HHLSCPNNLVVEDEG 56 76 117 357 HLSCPNNLVVEDEGC 65 119 111 358LSCPNNLVVEDEGCT 69 117 127 359 SCPNNLVVEDEGCTN 83 114 91 360CPNNLVVEDEGCTNL 77 97 49 361 PNNLVVEDEGCTNLS 78 107 97 362NNLVVEDEGCTNLSG 72 99 97 363 NLVVEDEGCTNLSGF 75 119 55 364LVVEDEGCTNLSGFS 76 103 52 365 VVEDEGCTNLSGFSY 73 107 91 366VEDEGCTNLSGFSYM 74 103 31 367 EDEGCTNLSGFSYME 54 90 7 368DEGCTNLSGFSYMEL 1 23 1 369 EGCTNLSGFSYMELK 51 114 129 370GCTNLSGFSYMELKV 55 114 118 371 CTNLSGFSYMELKVG 47 110 137 372TNLSGFSYMELKVGY 43 106 161 373 NLSGFSYMELKVGYI 61 115 170 374LSGFSYMELKVGYIL 71 132 169 375 SGFSYMELKVGYILA 79 132 161 376GFSYMELKVGYILAI 65 111 141 377 FSYMELKVGYILAIK 89 112 192 378SYMELKVGYILAIKM 65 123 152 379 YMELKVGYILAIKMN 78 114 150 380MELKVGYILAIKMNG 76 141 107 381 ELKVGYILAIKMNGF 87 132 76 382LKVGYLLAIKMNGFT 78 112 118 383 KVGYILAIKMNGFTC 78 118 68 384VGYLLAIKMNGFTCT 77 93 1 385 GYILAIKMNGFTCTG 75 90 1 386 YILAIKMNGFTCTGV47 107 107 387 ILAIKMNGFTCTGVV 79 103 104 388 LAIKMNGFTCTGVVT 68 130 159389 AIKMNGFTCTGVVTE 47 103 152 390 IKMNGFTCTGVVTEA 68 108 138 391KMNGFTCTGVVTEAE 76 104 133 392 MNGFTCTGVVTEAEN 69 99 148 393NGFTCTGVVTEAENY 69 101 138 394 GFTCTGVVTEAENYT 71 86 129 395FTCTGVVTEAENYTN 83 125 154 396 TCTGVVTEAENYTNF 92 112 129 397CTGVVTEAENYTNFV 76 123 150 398 TGVVTEAENYTNEVG 85 110 154 399GVVTFAFNYTNFVGY 86 111 110 400 VVTEAENYTNFVGYV 87 106 114 401VTEAENYTNFVGYVT 79 90 73 402 TEAENYTNFVGYVTT 68 84 8 403 EAENYTNFVGYVTTT69 117 142 404 AENYTNFVGYVTTTF 66 106 110 405 ENYTNFVGYVTTTFK 44 112 183406 NYTNFVGYVTTTFKR 49 114 174 407 YTNFVGYVTTTFKRK 51 104 138 408TNFVGYVTTTFKRKH 71 125 165 409 NFVGYVTTTFKRKHF 65 107 154 410FVGYVTTTFKRKHFR 70 111 152 411 VGYVTTTFKRKHFRP 75 113 155 412GYVTTTFKRKHFRPT 70 123 160 413 YVTTTFKRKHFRPTP 85 106 160 414VTTTFKRKHFRPTPD 79 105 119 415 TTTFKRKHFRPTPDA 80 108 137 416TTFKRKHFRPTPDAC 74 99 110 417 TFKRKHFRPTPDACR 96 111 108 418FKRKHERPTPDACRA 64 92 62 419 KRKHFRPTPDACRAA 65 93 1 420 RKHFRPTPDACRAAY64 107 99 421 KHFRPTPDACRAAYN 73 112 124 422 HFRPTPDACRAAYNW 46 113 118423 FRPTPDACRAAYNWK 43 112 148 424 RPTPDACRAAYNWKM 77 101 129 425PTPDACRAAYNWKMA 99 125 143 426 TPDACRAAYNWKMAG 92 132 160 427PDACRAAYNWKMAGD 61 124 147 428 DACRAAYNWKMAGDP 84 113 136 429ACRAAYNWKMAGDPR 82 116 138 430 CRAAYNWKMAGDPRY 87 118 137 431RAAYNWKMAGDPRYE 90 130 120 432 AAYNWKMAGDPRYEE 68 106 120 433AYNWKMAGDPRYEES 96 94 77 434 YNWKMAGDPRYEESL 83 118 116 435NWKMAGDPRYEESLH 58 101 69 436 WKMAGDPRYEESLHN 69 101 1 437KMAGDPRYEESLHNP 62 102 84 438 MAGDPRYEESLHNPY 64 116 112 439AGDPRYEESLHNPYP 40 101 125 440 GDPRYEESLHNPYPD 36 98 123 441DPRYEESLHNPYPDY 57 110 118 442 PRYEESLHNPYPDYR 73 115 129 443RYEESLHNPYPDYRW 69 112 125 444 YEESLHNPYPDYRWL 58 106 120 445EESLHNPYPDYRWLR 76 123 141 446 ESLHNPYPDYRWLRT 92 132 125 447SLHNPYPDYRWLRTV 78 111 137 448 LHNPYPDYRWLRTVK 79 106 142 449HNPYPDYRWLRTVKT 86 108 146 450 NPYPDYRWLRTVKTT 85 102 151 451PYPDYRWLRTVKTTK 65 93 103 452 YPDYRWLRTVKTTKE 72 97 97 453PDYRWLRTVKTTKES 76 85 27 454 DYRWLRTVKTTKESL 54 111 105 455YRWLRTVKTTKESLV 46 117 125 456 RWLRTVKTTKESLVI 40 110 120 457WLRTVKTTKESLVII 41 104 125 458 LRTVKTTKESLVIIS 65 104 161 459RTVKTTKESLVIISP 82 120 150 460 TVKTTKESLVIISPS 76 116 150 461VKTTKESLVIISPSV 71 120 154 462 KTTKESLVIISPSVA 101 112 147 463TTKESLVIISPSVAD 78 121 141 464 TKESLVIISPSVADL 86 112 132 465KESLVIISPSVADLD 86 117 111 466 ESLVIISPSVADLDP 88 125 143 467SLVIISPSVADLDPY 68 105 125 468 LVIISPSVADLDPYD 85 107 93 469VIISPSVADLDPYDR 59 98 50 470 IISPSVADLDPYDRS 83 125 14 471ISPSVADLDPYDRSL 50 119 91 472 SPSVADLDPYDRSLH 59 114 118 473PSVADLDPYDRSLHS 44 114 118 474 SVADLDPYDRSLHSR 49 106 129 475VADLDPYDRSLHSRV 71 113 141 476 ADLDPYDRSLHSRVF 70 121 141 477DLDPYDRSLHSRVFP 111 152 127 478 LDPYDRSLHSRVFPS 99 142 106 479DPYDRSLHSRVFPSG 90 120 134 480 PYDRSLHSRVFPSGK 86 120 130 481YDRSLHSRVFPSGKC 364 818 127 482 DRSLHSRVFPSGKCS 98 142 141 483RSLHSRVFPSGKCSG 87 141 156 484 SLHSRVFPSGKCSGV 69 111 141 485LHSRVFPSGKCSGVA 78 114 129 486 HSRVFPSGKCSGVAV 97 118 111 487SRVFPSGKCSGVAVS 100 125 24 488 RVFPSGKCSGVAVSS 69 110 106 489VFPSGKCSGVAVSST 74 114 142 490 FPSGKCSGVAVSSTY 64 134 146 491PSGKCSGVAVSSTYC 56 112 132 492 SGKCSGVAVSSTYCS 64 121 120 493GKCSGVAVSSTYCST 92 143 145 494 KCSGVAVSSTYCSTN 88 130 130 495CSGVAVSSTYCSTNH 110 165 143 496 SGVAVSSTYCSTNHD 79 110 115 497GVAVSSTYCSTNHDY 79 114 108 498 VAVSSTYCSTNHDYT 85 114 118 499AVSSTYCSTNHDYTI 71 105 102 500 VSSTYCSTNHDYTIW 78 107 121 501SSTYCSTNHDYTIWM 76 107 121 502 STYCSTNHDYTIWMP 86 99 119 503TYCSTNHDYTIWMPE 96 107 74 504 YCSTNHDYTIWMPEN 47 92 29 505CSTNHDYTIWMPENP 52 106 86 506 STNHDYTIWMPENPR 60 112 107 507TNHDYTIWMPENPRL 69 129 119 508 NHDYTIWMPENPRLG 71 119 130 509HDYTIWMPENPRLGM 82 125 123 510 DYTIWMPENPRLGMS 93 127 123 511YTIWMPENPRLGMSC 97 132 143 512 TIWMPENPRLGMSCD 69 106 134 513IWMPENPRLGMSCDI 98 110 101 514 WMPENPRLGMSCDIF 88 113 120 515MPENPRLGMSCDIFT 105 121 143 516 PENPRLGMSCDIFTN 83 111 104 517ENPRLGMSCDLFTNS 71 118 111 518 NPRLGMSCDIFTNSR 90 113 138 519PRLGMSCDIFTNSRG 72 112 105 520 RLGMSCDIFTNSRGK 88 106 113 521LGMSCDIFTNSRGKR 76 110 114 522 GMSCDIFTNSRGKRA 54 120 101 523MSCDLFTNSRGKRAS 46 110 106 524 SCDIFTNSRGKRASK 44 111 98 525CDLFTNSRGKRASKG 42 104 117 526 DIFTNSRGKRASKGS 70 107 111 527IFTNSRGKRASKGSE 77 125 87 528 FTNSRGKRASKGSET 83 111 119 529TNSRGKRASKGSETC 68 108 110 530 NSRGKRASKGSETCG 92 100 119 531SRGKRASKGSETCGF 64 93 90 532 RGKRASKGSETCGFV 75 104 115 533GKRASKGSETCGFVD 92 124 118 534 KRASKGSETCGFVDE 92 106 129 535RASKGSETCGFVDER 86 110 134 536 ASKGSETCGFVDERG 97 108 103 537SKGSETCGFVDERGL 92 102 76 538 KGSETCGFVDERGLY 90 97 44 539GSETCGFVDERGLYK 57 115 92 540 SETCGFVDERGLYKS 33 116 86 541ETCGFVDERGLYKSL 64 120 138 542 TCGFVDERGLYKSLK 47 120 125 543CGFVDERGLYKSLKG 72 115 120 544 GFVDERGLYKSLKGA 84 120 129 545FVDERGLYKSLKGAC 86 121 124 546 VDERGLYKSLKGACK 50 108 110 547DERGLYKSLKGACKL 90 119 54 548 ERGLYKSLKGACKLK 90 118 106 549RGLYKSLKGACKLKL 90 121 121 550 GLYKSLKGACKLKLC 94 129 92 551LYKSLKGACKLKLCG 93 136 141 552 YKSLKGACKLKLCGV 80 112 110 553KSLKGACKLKLCGVL 129 113 110 554 SLKGACKLKLCGVLG LKGACKLKLCGVLGLKGACKLKLCGVLGLR GACKLKLCGVLGLRL ACKLKLCGVLGLRLM CKLKLCGVLGLRLMDKLKLCGVLGLRLMDG LKLCGVLGLRLMDGT KLCGVLGLRLMDGTW

111 90 111 134 117 111 120 145 132 124 23 100 129 142 147 114 148 86 314315 316 317 318 319 320 321 322 LCGVLGLRLMDGTWV 83 138 129 555CGVLGLRLMDGTWVA 99 117 104 556 GVLGLRLMDGTWVAM 89 148 117 557VLGLRLMDGTWVAMQ 90 141 127 558 LGLRLMDGTWVAMQT 102 115 97 559GLRLMDGTWVAMQTS 104 138 120 560 LRLMDGTWVAMQTSN 103 114 118 561RLMDGTWVAMQTSNE 100 113 130 562 LMDGTWVAMQTSNET 96 106 106 563MDGTWVAMQTSNETK 97 97 110 564 DGTWVAMQTSNETKW 69 114 92 565GTWVAMQTSNETKWC 58 113 82 566 TWVAMQTSNETKWCP 78 118 107 567WVAMQTSNETKWCPP 50 114 116 568 VAMQTSNETKWCPPD 86 104 151 569AMQTSNETKWCPPDQ 104 114 128 570 MQTSNETKWCPPDQL 104 132 125 571QTSNETKWCPPDQLV 92 120 155 572 TSNETKWCPPDQLVN 97 111 90 573SNETKWCPPDQLVNL 99 129 110 574 NETKWCPPDQLVNLH 90 128 107 575ETKWCPPDQLVNLHD 105 120 118 576 TKWCPPDQLVNLHDF 85 125 125 577KWCPPDQLVNLHDFR 89 113 121 578 WCPPDQLVNLHDFRS 101 119 99 579CPPDQLVNLHDFRSD 93 137 127 580 PPDQLVNLHDFRSDE 107 120 56 581PDQLVNLHDFRSDEI 35 106 63 582 DQLVNLHIDFRSDEW 54 117 97 583QLVNLHDFRSDEIEH 60 113 106 584 LVNLHDFRSDEIEHL 47 104 100 585VNLHDFRSDEIEHLV 83 129 98 586 NLHDFRSDEIEHLVV 83 113 110 587LHDFRSDEIEHLVVE 93 115 121 588 HDFRSDEIEHLVVEE 69 107 150 589DFRSDEIEHLVVEEL 99 103 110 590 FRSDEIEHIVVEELV 86 114 116 591RSDEIEHLVVEELVR 100 138 104 592 SDEIEHLVVEELVRK 101 117 118 593DEIEHLVVEELVRKR 94 123 143 594 EIEHLVVEELVRKRE 82 113 121 595IEHLVVEELVRKREE 90 129 118 596 EHLVVEELVRKREEC 82 114 106 597HLVVEELVRKREECL 82 123 46 598 LVVEELVRKREECLD 64 100 79 599VVEELVRKREECLDA 62 108 97 600 VEELVRKREECLDAL 58 111 101 601EELVRKREECLDALE 69 112 123 602 ELVRKREECLDALES 82 113 117 603LVRKREECLDALESI 86 130 124 604 VRKREECLDALESIM 58 181 151 605RKREECLDALESIMT 73 110 137 606 KREECLDALESIMTT 102 113 97 607REECLDALESIMTTK 94 110 106 608 EECLDALESIMTTKS 82 120 133 609ECLDALESIMTTKSV 91 112 125 610 CLDALESIMTTKSVS 101 146 155 611LDALESIMTTKSVSF 97 116 152 612 DALESIMTTKSVSFR 104 120 188 613ALESIMTTKSVSFRR 97 132 137 614 LESIMTTKSVSFRRL 48 114 130 615ESIMTTKSVSFRRLS 62 111 114 616 SIMTTKSVSFRRLSH 54 130 97 617IMTTKSVSFRRLSHL 43 101 111 618 MTTKSVSFRRLSHLR 59 116 125 619TTKSVSFRRLSHLRK 66 118 111 620 TKSVSFRRLSHLRKL 83 125 123 621KSVSFRRLSHLRKLV 108 124 129 622 SVSFRRLSHLRKLVP 64 123 117 623VSFRRLSHLRKLVPG 90 111 105 624 SFRRLSHLRKLVPGF 92 110 96 625FRRLSHLRKLVPGFG 90 108 111 626 RRLSHLRKLVPGFGK 92 143 118 627RLSHLRKLVPGFGKA 93 123 148 628 LSHLRKLVPGFGKAY 96 139 150 629SHLRKLVPGFGKAYT 113 132 132 630 HLRKLVPGFGKAYTI 99 111 102 631LRKLVPGFGKAYTLF 83 118 82 632 RKLVPGFGKAYTIFN 47 115 86 633KLVPGFGKAYTIFNK 47 114 123 634 LVPGFGKAYTLFNKT 54 112 139 635VPGFGKAYTIFNKTL 58 114 138 636 PGFGKAYTIFNKTLM 78 113 157 637GFGKAYTIFNKTLME FGKAYTIFNKTLMEA GKAYTIFNKTLMEAD KAYTIFNKTLMEADAAYTIFNKTLMEADAH YTIFNKTLMEADAHY TIFNKTLMEADAHYK IFNKTLMEADAHYKSFNKTLMEADAHYKSV 78 90 76 101 86 104 107 100 111 123 151 127 123 121 147123 118 141

323 324 325 326 327 328 329 330 331 NKTLMEADAHYKSVR 104 116 141 638KTLMEADAHYKSVRT 91 98 123 639 TLMEADAHYKSVRTW 100 114 90 640LMEADAHYKSVRTWN 73 107 97 641 MEADAHYKSVRTWNE 62 129 83 642EADAHYKSVRTWNEI 58 97 106 643 ADAHYKSVRTWNEIL 56 100 100 644DAHYKSVRTWNEILP 59 121 112 645 AHYKSVRTWNEILPS 112 160 125 646HYKSVRTWNEILPSK 80 130 123 647 YKSVRTWNE1LPSKG 66 137 116 648KSVRTWNEILPSKGC 115 125 114 649 SVRTWNEILPSKGCL 106 138 118 650VRTWNEILPSKGCLR 90 124 133 651 RTWNEILPSKGCLRV 120 127 120 652TWNEILPSKGCLRVG 97 146 127 653 WNEILPSKGCLRVGG 102 136 117 654NEILPSKGCLRVGGR 104 130 163 655 EILPSKGCLRVGGRC 104 112 128 656ILPSKGCLRVGGRCH 79 113 107 657 LPSKGCLRVGGRCHP 77 119 100 658PSKGCLRVGGRCHPH 69 138 91 659 SKGCLRVGGRCHPHV 72 121 103 660KGCLRVGGRCHPHVN 68 130 115 661 GCLRVGGRCHPHVNG 85 125 123 662CLRVGGRCHPHVNGV 102 132 134 663 LRVGGRCHPHVNGVF 104 143 133 664RVGGRCHPHVNGVFF 86 143 99 665 VGGRCHPHVNGVFFN 120 136 120 666GGRCHPHVNGVFFNG 86 119 119 667 GRCHPHVNGVFFNGI 117 113 117 668RCHPHVNGVFFNGII 98 141 143 669 CHPHVNGVFFNGIIL 97 150 151 670HPHVNGVFFNGIILG 104 138 164 671 PHVNGVFFNGLILGP 93 173 146 672HVNGVFFNGIILGPD 97 123 114 673 VNGVFFNGIILGPDG 68 116 85 674NGVFFNGIILGPDGN 66 117 97 675 GVFFNGIILGPDGNV 58 116 100 676VFFNGIILGPDGNVL 55 132 108 677 FFNGIILGPDGNVLI 92 143 105 678FNGIILGPDGNVLIP 61 139 130 679 NGIILGPDGNVLTPE 102 146 141 680GIILGPDGNVLTPEM 107 132 123 681 IILGPDGNVLIPEMQ 85 118 93 682ILGPDGNVLIPEMQS 125 134 119 683 LGPDGNVLIPEMQSS 100 134 150 684GPDGNVLIPEMQSSL 86 154 157 685 PDGNVLIPEMQSSLL 87 129 139 686DGNVLIPEMQSSLLQ 123 134 169 687 GNVLIPEMQSSLLQQ 96 120 168 688NVLIPEMQSSLLQQH 72 120 150 689 VLIPEMQSSLLQQHM 92 104 142 690LIPEMQSSLLQQHME 89 111 85 691 IPEMQSSLLQQHMEL 89 128 129 692PEMQSSLLQQHMELL 62 133 93 693 EMQSSLLQQHMELLE 58 129 142 694MQSSLLQQHMELLES 65 113 117 695 QSSLLQQHMELLESS 82 114 132 696SSLLQQHMELLESSV 90 128 132 697 SLLQQHMELLESSVI 124 163 133 698LLQQHMELLESSVIP 78 111 121 699 LQQHMELLESSVIPL 106 134 128 700QQHMELLESSVIPLV 103 134 133 701 QHMELLESSVLPLVH 98 146 139 702HMELLESSVIPLVHP 110 129 134 703 MELLESSVIPLVHPL 90 125 152 704ELLESSVLPLVHPLA 90 133 155 705 LLESSVIPLVHPLAD 72 117 118 706LESSVLPLVHPLADP 90 128 128 707 ESSVIPLVHPLADPS 104 138 143 708SSVIPLVHPLADPST 73 104 93 709 SVIPLVHPLADPSTV 72 137 107 710VIPLVHPLADPSTVF 69 141 123 711 IPLVHPLADPSTVFK 96 156 188 712PLVHPLADPSTVFKD 93 112 138 713 LVHPLADPSTVFKDG 164 174 188 714VHPLADPSTVFKDGD 98 138 125 715 HPLADPSTVFKDGDE 74 141 117 716PLADPSTVFKDGDEA 99 125 90 717 LADPSTVFKDGDEAE 68 116 113 718ADPSTVFKDGDEAED 147 152 110 719 DPSTVFKDGDEAEDF 98 147 137 720PSTVFKDGDEAEDFV 104 143 141 721 STVFKDGDEAEDFVE 104 120 125 722TVFKDGDEAEDFVEV 107 124 96 723 VFKDGDEAEDFVEVH 100 106 93 724FKDGDEAEDFVEVHL 65 76 119 725 KDGDEAEDFVEVHLP 72 93 76 726DGDEAEDFVEVHLPD 85 123 91 727 GDEAEDFVEVHLPDV 46 124 113 728DEAEDFVEVHLPDVH 68 136 123 729 EAEDVFEVHLPDVHN 76 117 114 730AEDFVEVHLPDVHNQ 123 138 123 731 EDFVEVHLPDVHNQV 90 141 123 732DFVEVHLPDVHNQVS 96 141 118 733 FVEVHLPDVHNQVSG 92 143 102 734VEVHLPDVHNQVSGV 106 141 123 735 EVHLPDVHNQVSGVD 91 150 139 736VHLPDVHNQVSGVDL 110 114 137 737 HLPDVHNQVSGVDLG 104 150 129 738LPDVHNQVSGVDLGL 104 154 154 739 PDVHNQVSGVDLGLP 106 129 115 740DVHNQVSGVDLGLPN 117 133 113 741 VHNQVSGVDLGLPNW 100 119 38 742HNQVSGVDLGLPNWG 76 106 38 743 NQVSGVDLGLPNWGK 78 138 98 744 Average 91.9119.5 130.9 StDV 157.9 37.6 169.8

TABLE 15 Neutralizing potencies of anti-rabies virus G protein IgGs.Name IgG IU/mg CR04-001 89 CR04-004 5 CR04-008 1176 CR04-010 3000CR04-018 1604 CR04-021 1500 CR04-026 <2 CR04-031 272 CR04-038 2330CR04-040 3041 CR04-060 89 CR04-073 6116 CR04-097 <1 CR04-098 7317CR04-103 3303 CR04-104 4871 CR04-108 4871 CR04-120 4938 CR04-125 4718CR04-126 2655 CR04-140 478 CR04-144 6250 CR04-146 ND CR04-164 4724 CR573800 CRJB 605 ND = not determined

TABLE 16 Neutralizing potencies of anti-rabies virus G protein IgGsagainst E57 escape viruses. E57A2 E57A3 E57B1 E57B2 E57B3 E57C3 Name IgG(IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg) CR04-008   0* 0 0 0 0 0CR04-010 8127 1355 5418 1355 2709 4064 CR04-018 1604 0 1604 0 59 535CR04-021  450 2 150 8 50 50 CR04-038 9437 1573 9437 1049 6291 1573CR04-040 8209 2736 24628 1368 5473 912 CR04-073 8256 1835 11008 18353669 1835 CR04-098 9878 3293 9878 3293 3293 3293 CR04-103 8917 297217835 2972 5945 2972 CR04-104 3288 2192 6576 2192 4384 1096 CR04-1083288 731 4384 731 2192 731 CR04-120 1111 14 741 82 247 41 CR04-125  70839 236 79 157 79 CR04-126  88 0 18 0 18 0 CR04-144 5625 2813 11250 28135625 1875 CR04-164 4252 472 4252 472 945 709 *0 indicates no 50%endpoint at a dilution of 1:100 of the antibody

TABLE 17 Neutralizing potency of CR-57 against E98 escape viruses. E98-2E98-4 E98-5 E98-6 E98-7 (IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg) CR-572813 8438 4219 2813 8438 CR04-098 0* 0 0 0 0 *Zero indicates no 50%endpoint at a dilution of 1:1000 of the antibody.

TABLE 18 Occurrence of amino acid residues in the minimal binding regionof CR57 within genotype 1 rabies viruses. K L C G V L Wild- K L C G(98.7%) V (99.6%) L (70.7%) type (99.6%)* (100%) (100%) R (0.4%) E(0.9%) I (0.4%) P (26.7%) R (0.4%) S (2.6%) *Percentage of occurrence ofeach amino acid is shown within 229 rabies virus isolates.

REFERENCES

-   Ameyama S., H. Toriumi, T. Takahashi, Y. Shimura, T. Nakahara, Y.    Honda, K. Mifune, T. Uchiyama and A. Kawai (2003). Monoclonal    antibody #3-9-16 recognizes one of the two isoforms of rabies virus    matrix protein that exposes its N-terminus on the virion surface.    Microbiol. Immunol. 47:639-651.-   Badrane H. and N. Tordo (2001). Host switching in Lyssavirus history    from the Chiroptera to the Carnivora orders. J. Virol. 75:8096-8104.-   Benmansour A., H. Leblois, P. Coulon, C. Tuffereau, Y. Gaudin, A.    Flamand and F. Lafay (1991). Antigenicity of rabies virus    glycoprotein. J. Virol. 65:4198-4203.-   Boel E., S. Verlaan, M. J. Poppelier, N. A. Westerdaal, J. A. Van    Strijp and T. Logtenberg (2000). Functional human monoclonal    antibodies of all isotypes constructed from phage display    library-derived single-chain Fv antibody fragments. J. Immunol.    Methods 239:153-166.-   Bunschoten H., M. Gore, I. J. Claassen, F. G. Uytdehaag, B.    Dietzschold, W. H. Wunner and A. D. Osterhaus (1989).    Characterization of a new virus-neutralizing epitope that denotes a    sequential determinant on the rabies virus glycoprotein. J. Gen.    Virol. 70 (Pt 2):291-8.-   Burton D. R. and C. F. Barbas (1994). Human antibodies from    combinatorial libraries. Adv. Immunol. 57:191-280.-   Champion J. M., R. B. Kean, C. E. Rupprecht, A. L. Notkins, H.    Koprowski, B. Dietzschold and D. C. Hooper (2000). The development    of monoclonal human rabies virus-neutralizing antibodies as a    substitute for pooled human immune globulin in the prophylactic    treatment of rabies virus exposure. J. Immunol. Methods 235:81-90.-   Chou T. C. and P. Talalay (1984). Quantitative analysis of    dose-effect relationships: the combined effects of multiple drugs or    enzyme inhibitors. Adv. Enzyme Regul. 22:27-55.-   Coulon P., J. P. Temaux, A. Flamand and C. Tuffereau (1998). An    avirulent mutant of rabies virus is unable to infect motoneurons in    vivo and in vitro. J. Virol. 72:273-278.-   De Kruif J., L. Terstappen, E. Boel and T. Logtenberg (1995a). Rapid    selection of cell subpopulation-specific human monoclonal antibodies    from a synthetic phage antibody library. Proc. Natl. Acad. Sci. USA    92:3938.-   De Kruif J., E. Boel and T. Logtenberg (1995b). Selection and    application of human single-chain Fv antibody fragments from a    semi-synthetic phage antibody display library with designed CDR3    regions. J. Mol. Biol. 248:97-105.-   Dietzschold B., W. H. Wunner, T. J. Wiktor, A. D. Lopes, M.    Lafon, C. L. Smith and H. Koprowski (1983). Characterization of an    antigenic determinant of the glycoprotein that correlates with    pathogenicity of rabies virus. Proc. Natl. Acad. Sci. USA 80:70-74.-   Dietzschold B., M. Gore, P. Casali, Y. Ueki, C. E. Rupprecht, A. L.    Notkins and H. Koprowski (1990). Biological characterization of    human monoclonal antibodies to rabies virus. J. Virol. 64:3087-3090.-   Hanlon C. A., C. A. DeMattos, C. C. DeMattos, M. Niezgoda, D. C.    Hooper, H. Koprowski, A. Notkins and C. E. Rupprecht (2001).    Experimental utility of rabies virus-neutralizing human monoclonal    antibodies in post-exposure prophylaxis. Vaccine 19:3834-3842.-   Huls G., I. J. Heijnen, E. Cuomo, J. van der Linden, E. Boel, J. van    de Winkel and T. Logtenberg (1999). Antitumor immune effector    mechanisms recruited by phage display-derived fully human IgG1 and    IgA1 monoclonal antibodies. Cancer Res. 59:5778-5784.-   Jones D., N. Kroos, R. Anema, B. van Montfort, A. Vooys, S. van der    Kraats, E. van der Helm, S. Smits, J. Schouten, K. Brouwer, F.    Lagerwerf, P. van Berkel, D. J. Opstelten, T. Logtenberg and A. Bout    (2003). High-level expression of recombinant IgG in the human cell    line PER.C6. Biotechnol. Prog. 19:163-168.-   Lafon M., T. J. Wiktor and R. I. Macfarlan (1983). Antigenic sites    on the CVS rabies virus glycoprotein: analysis with monoclonal    antibodies. J. Gen. Virol. 64 (Pt 4):843-851.-   Luo T. R., N. Minamoto, H. Ito, H. Goto, S. Hiraga, N. Ito, M.    Sugiyama and T. Kinjo (1997). A virus-neutralizing epitope on the    glycoprotein of rabies virus that contains Trp251 is a linear    epitope. Virus Res. 51:35-41.-   Madhusudana S. N., R. Shamsundar and S. Seetharaman (2004). In vitro    inactivation of the rabies virus by ascorbic acid. Int. J. Infect.    Dis. 8:21-25.-   Ni Y., Y. Tominaga, Y. Honda, K. Morimoto, S. Sakamoto and A. Kawai    (1995). Mapping and characterization of a sequential epitope on the    rabies virus glycoprotein which is recognized by a neutralizing    monoclonal antibody, RG719. Microbiol. Immunol. 39:693-702.-   Prehaud C., P. Coulon, F. LaFay, C. Thiers and A. Flamand (1988).    Antigenic site II of the rabies virus glycoprotein:structure and    role in viral virulence. J. Virol. 62:1-7.-   Schumacher C. L., B. Dietzschold, H. C. Ertl, H. S, Niu, C. E.    Rupprecht and H. Koprowski (1989). Use of mouse anti-rabies    monoclonal antibodies in post-exposure treatment of rabies. J. Clin.    Invest. 84:971-975.-   Seif I., P. Coulon, E. Rollin and A. Flamand (1985). Rabies    virulence: effect on pathogenicity and sequence characterization of    rabies virus mutations affecting antigenic site III of the    glycoprotein. J. Virol. 53:926-934.-   Slootstra J. W., W. C. Puijk, G. J. Ligtvoet, J. P. Langeveld    and R. H. Meloen (1996). Structural aspects of antibody-antigen    interaction revealed through small random peptide libraries. Mol.    Divers. 1:87-96.-   Tordo N. (1996). Characteristics and molecular biology of rabies    virus. In F. -X. Meslin, M. M. Kaplan, H. Koprowski, editors,    Laboratory Techniques in rabies, 4^(th) edition Geneva, Switzerland:    World Health Organization.-   White L. A. and W. A. Chappell (1982). Inactivation of rabies virus    in reagents used for the fluorescent rabies antibody test. J. Clin.    Microbiol. 16:253-256.

1. A pharmaceutical composition comprising at least two rabies virusneutralizing monoclonal antibodies or antigen-binding fragments thereof,that are capable of reacting with different, non-competing epitopes ofthe rabies virus, wherein a first rabies virus neutralizing monoclonalantibody or antigen-binding fragment thereof, is capable of reactingwith an epitope located in antigenic site I wherein the epitopecomprises amino acids 226-231 of the rabies virus G protein and a secondrabies virus neutralizing monoclonal antibody, or antigen-bindingfragment thereof, is capable of reacting with an epitope located inantigenic site III of the rabies virus G protein.
 2. A pharmaceuticalcomposition comprising two rabies virus neutralizing binding moleculesthat are capable of binding to different, non-competing epitopes of therabies virus, the composition comprising: a first rabies virusneutralizing binding molecule capable of binding to an epitope locatedin antigenic site I of the rabies virus G protein, said first rabiesvirus neutralizing antibody comprising: a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 273 or a sequence thatis at least 80% homologous thereto, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 275 or a sequence thatis at least 80% homologous thereto, and a second rabies virusneutralizing binding molecule capable of binding to an epitope locatedin antigenic site III of the rabies virus G protein.
 3. Thepharmaceutical composition of claim 2, wherein the second rabies virusneutralizing binding molecule comprises: i) a heavy chain variableregion comprising SEQ ID NO: 29 and a light chain variable regioncomprising SEQ ID NO: 53; or ii) a heavy chain variable regioncomprising SEQ ID NO: 35 and a light chain variable region comprisingSEQ ID NO: 59; or iii) a heavy chain variable region comprising SEQ IDNO: 39 and a light chain variable region comprising SEQ ID NO: 63; oriv) a heavy chain variable region comprising SEQ ID NO: 40 and a lightchain variable region comprising SEQ ID NO: 64; or v) a heavy chainvariable region comprising SEQ ID NO: 41 and a light chain variableregion comprising SEQ ID NO: 65; or vi) a heavy chain variable regioncomprising SEQ ID NO: 47 and a light chain variable region comprisingSEQ ID NO:
 71. 4. The pharmaceutical composition of claim 2, wherein thefirst rabies virus binding molecule is an IgG and the second rabiesvirus binding molecule is an IgG.
 5. A pharmaceutical compositioncomprising at least two rabies virus neutralizing monoclonal antibodies,or antigen-binding fragments thereof, that are able to react withdifferent, non-competing epitopes of rabies virus, wherein a firstrabies virus neutralizing monoclonal antibody or antigen-bindingfragment thereof, is able to react with an epitope located in antigenicsite I and comprising amino acids 226-231 of the rabies virus G protein,and a second rabies virus neutralizing monoclonal antibody orantigen-binding fragment thereof, is able to react with an epitopelocated in antigenic site III of the rabies virus G protein, whereinsaid rabies virus neutralizing antibodies are human antibodies.